Hybridization Chain Reaction FISH in the Developing Drosophila Optic Lobe

Summary

This protocol describes the steps required to perform hybridization chain reaction fluorescent in situ hybridization (HCR-FISH) in Drosophila melanogaster larval optic lobes. Procedures for probe design, hybridization, amplification, and tissue mounting are detailed to facilitate visualization of the developing optic lobe.

Abstract

Understanding neural development requires precise characterization of when and where genes are expressed. Techniques such as in situ hybridization have long enabled visualization of gene expression in developing tissues. Although in situ hybridization is widely used in Drosophila melanogaster embryos, its application to visual system development has been limited by several technical challenges. For example, long probes often penetrate poorly into the optic lobe, and protocols that rely on diffusible catalytic reporters, such as horseradish peroxidase, often yield low signal-to-noise ratios in whole-mount preparations. Hybridization chain reaction fluorescent in situ hybridization (HCR-FISH) overcomes many of these limitations and enables more robust signal detection. As in conventional FISH, DNA probe monomers hybridize to RNA sequences within tissues. In HCR-FISH, however, fluorescently conjugated amplifier probes form stable hairpin structures that trigger polymerization of additional monomers, thereby amplifying signal intensity. The use of multiplexed probe sets also permits the use of shorter oligonucleotides (~45 nt, compared with up to 1,000 nt in conventional FISH), improving probe penetration and producing more uniform staining in whole-mount samples. This article describes an HCR-FISH protocol optimized for the Drosophila larval optic lobe. The protocol adapts existing HCR-FISH approaches by optimizing fixation conditions and increasing probe and hairpin concentrations to improve signal intensity and tissue penetration. Reagent lists and step-by-step procedures are provided. Potential applications in developmental neurobiology are also presented, including visualization of newborn neurons, and characterization of gene expression patterns in cases where conventional methods, such as endogenously tagged proteins or antibody staining, fail to provide sufficient information.

Introduction

Understanding how the brain is assembled requires identifying both the diversity of cell types present and the gene repertoires expressed within each cell type¹. Recent advances in single-cell RNA sequencing have enabled researchers to address these questions at high resolution². However, many single-cell approaches do not preserve spatial information, requiring additional experiments to map transcriptional cell-type clusters to their locations within the developing brain.

Binary gene expression systems such as GAL4-UAS can provide positional information but are not quantitative³. In addition, delays between gene expression and reporter detection can obscure physiologically relevant expression patterns during early developmental stages. Immunostaining is another widely used approach for determining tissue-specific expression patterns⁴. However, secreted proteins are often diffuse and difficult to localize accurately.

Nucleotide-labeling approaches, such as in situ hybridization (ISH), have also been widely used to obtain spatial information about gene expression⁵. In ISH, labeled DNA oligonucleotides bind to reverse-complementary RNA sequences of interest. Early ISH methods used radiolabeled DNA probes⁶, whereas later protocols incorporated safer labels such as digoxigenin, fluorescein, and rhodamine^7,8,9. Scientists rapidly adapted ISH for use in Drosophila melanogaster, where it was applied to characterize polytene chromosome organization and map the expression patterns of gap genes such as hunchback in whole-mount embryos^10,11.

Despite its widespread utility, several technical limitations hindered the adoption of ISH in thicker whole-mount preparations such as the larval optic lobe. Many in situ protocols relied on diffusible catalytic reporters, such as horseradish peroxidase, which often resulted in low signal-to-noise ratios¹². In addition, many approaches used long probes targeting single sequences to improve specificity. Although effective in Drosophila embryos and polytene chromosomes, long probes penetrate poorly into the larval brain and often produce uneven labeling^10,11,13.

The development of single-molecule fluorescent ISH (smFISH) and hybridization chain reaction fluorescent in situ hybridization (HCR-FISH) addressed many of these limitations^14,15. By using sets of short probes tiled across a single gene region, smFISH reduces average probe length from approximately 1,000 nt to approximately 40 nt¹⁶. This substantially improves probe penetration while maintaining strong signal intensity. HCR-FISH further improves signal-to-noise ratios by using fluorescently conjugated amplifier probes that increase positional accuracy and amplify signal output. Importantly, this amplification step appears to scale linearly with transcript concentration, enabling quantitative measurement of gene expression levels¹⁷.

HCR-FISH begins with initiator probes complementary to the target sequence. These unlabeled DNA oligonucleotides contain a single-stranded initiator sequence that binds to the input domain of a fluorescently labeled H1 amplifier probe. H1 amplifiers adopt a hairpin structure that maintains catalytic inactivity until binding to the initiator sequence. Upon binding, the H1 amplifier opens and exposes a sequence that binds to a partner probe, H2. This interaction opens the H2 hairpin, allowing it to bind another H1 probe (Figure 1)¹⁵. Repeated cycles of H1/H2 binding generate a stable polymer that amplifies the fluorescence signal while reducing background noise. Because each initiator sequence is specific to a corresponding H1/H2 pair, multiple probes with distinct initiator sequences and fluorophores can be used simultaneously to label several genes within the same sample.

Figure 1: Graphical representation of the HCR-FISH workflow. DNA probes containing initiator sequences hybridize to a target RNA transcript. Fluorescently labeled DNA amplifier hairpins bind to the initiator sequence and undergo sequential polymerization to form stable double-stranded amplification chains. Direct fluorophore conjugation improves signal amplification and signal-to-noise ratio. Please click here to view a larger version of this figure.

Recent improvements to HCR-FISH have further increased its utility. Split initiator probe design improves amplifier specificity, and secondary antibodies conjugated to HCR initiators enable simultaneous immunohistochemistry and HCR-FISH¹⁸. In addition, expanded fluorophore options now permit multiplexed imaging of up to 10 genes simultaneously^19,20.

Although initial proof-of-concept experiments for HCR-FISH were performed in zebrafish (Danio rerio), the method has gained widespread use across many systems, particularly in non-model organisms where antibodies are expensive or unavailable¹⁵. Probe synthesis also offers a faster alternative to antibody generation, which can require months of development. Consequently, HCR-FISH provides a rapid approach for validating candidate genes identified through single-cell RNA sequencing datasets²¹. Even in established genetic model organisms, HCR-FISH provides a valuable method for spatially mapping newly born neurons when GAL4 reporters or immunohistochemistry fail to adequately label specific cell populations.

This article describes an HCR-FISH protocol optimized for the third instar larval Drosophila optic lobe. The protocol adapts methods previously published in other organisms for use in the Drosophila visual system²². Fixation conditions commonly used for Drosophila immunohistochemistry were optimized for FISH applications. Probe and hairpin concentrations were increased to improve signal intensity and tissue penetration. PCR tubes were used instead of spot plates to reduce reagent consumption and simplify handling of small tissue samples. In addition, wash and hybridization times were shortened to accommodate the smaller size of the Drosophila brain relative to other insect systems commonly used for HCR-FISH.

Representative applications demonstrate how this approach can provide positional information for specific neuronal populations during development. These experiments indicate that certain transcripts, such as abrupt, can be detected earlier with HCR-FISH than with antibody staining or reporter lines. Potential challenges, troubleshooting strategies, and applications of HCR-FISH for studying visual system development in Drosophila are also discussed.

Protocol

All Drosophila melanogaster experiments were performed in accordance with the City College of New York, Columbia University, and New York University guidelines for the care and use of research organisms. The research tools used in this protocol are listed in the Table of Materials.

1. Prepare buffers

NOTE: Prepare all reagents in nuclease-free tubes using filter tips.
NOTE: Wear gloves throughout the procedure. Clean work surfaces with RNase decontamination solution before beginning.

1. Prepare nuclease-free solutions
   1. Prepare 1x PBS-T by diluting 20x PBS, Triton X-100, and glycine in nuclease-free water to final concentrations of 1x PBS, 1% (v/v) Triton X-100, and 1 mM glycine.
   2. Prepare 5x SSCT by diluting 20x SSC and Tween-20 in nuclease-free water to prepare 5x SSC containing 0.1% (v/v) Tween-20.
   3. Dilute 20x PBS in nuclease-free water to prepare 1x PBS.

2. Design probes

1. To design probes commercially, select the isoform of interest for the species of interest and order probes matched to the desired amplifier using the vendor's web interface.
   NOTE: Commercial HCR probe chemistries and initiator sequences are proprietary and under patent by Molecular Instruments.
   1. Modify the target region in the web interface if transcript-specific or pre-mRNA detection is desired.
   2. Order approximately 20 probes for highly expressed transcripts. Use up to 40 probes for low-abundance transcripts in whole-mount Drosophila preparations.
      NOTE: Order specialized oligos for shorter transcripts that accommodate fewer probe-binding sites.
2. To design probes manually, download the FASTA sequence for the target transcript. Determine whether to target a single isoform or all isoforms of the gene.
   NOTE: Prioritize constitutive exons or highly expressed isoforms identified from RNA sequencing datasets to improve signal.
   1. If designing probes for pre-mRNA detection, design probes spanning intron-exon junctions. Design probes spanning splice junctions to detect mature transcripts. Use intronic probes to detect nascent transcripts within nuclei.
      NOTE: Intronic probes may not colocalize with mature cytoplasmic transcripts and may require additional markers for cell identification.
   2. Determine probe set size. Use a minimum of approximately 5 probes for short transcripts when larger probe sets cannot be designed. Use 20 probes for standard applications and approximately 40 probes for optimal sensitivity in whole-mount samples^18,23.
   3. If designing your own probes, use 25nt sequences separated by a 2bp spacer and add split initiator sequences to the 5’ end of each oligo.
      NOTE: Although first-generation split initiator sequences are publicly available, next-generation commercial initiator sequences are proprietary¹⁸.
   4. If designing your own probes, validate the probe sequences. Ensure that probes within each set have similar melting temperatures. Confirm 100% identity between probes.
      NOTE: Avoid strong G-rich regions and maintain similar GC content among probes²⁴. Minimize repetitive sequences and evaluate off-target binding using BLAST²⁵.
   5. Use publicly available probe-design software when appropriate^26,27.
      NOTE: Open-source design tools may not reproduce the optimized performance of commercial probe-design pipelines.

3. Dissect, fix, and prehybridize brains

1. Prewarm Probe Hybridization Buffer to 37 °C.
   CAUTION: Probe Hybridization Buffer contains formamide. Handle under a chemical fume hood while wearing appropriate personal protective equipment.
2. Dilute each probe set to a final concentration of 1 µM using nuclease-free distilled water.
3. Aliquot approximately 250 µL of nuclease-free PBS onto a dissecting dish.
4. Transfer a wandering third instar larva into the PBS using forceps. Hold the abdomen with one pair of forceps and grasp the mouth hooks with a second pair. Pull the mouth hooks away from the body to isolate the brain and ventral nerve cord (VNC).
5. Remove fat bodies and salivary glands while retaining the VNC. Transfer dissected brains into a tube containing 600 µL of ice-cold nuclease-free PBS.
   NOTE: Keep optic lobes submerged throughout the dissection to prevent tissue drying.
6. Repeat dissection until approximately six optic lobes have been collected. Complete dissections within 20 min to minimize tissue degradation.
7. Add 200 µL of 16% paraformaldehyde (PFA) to achieve a final concentration of 4% PFA. Invert the tube gently to mix and incubate for 15 min at room temperature.
   CAUTION: PFA is toxic and mutagenic. Handle under a chemical fume hood while wearing gloves.
8. Remove fixative from the samples and deposit as designated chemical waste. Wash samples three times with 600 µL of cold nuclease-free PBS, using a dissecting dish to catch the wash solutions.
   NOTE: Inspect wash solutions for tissue loss and recover any displaced brains under a dissecting microscope.
9. Transfer brains into a PCR tube containing 100 µL of prewarmed Probe Hybridization Buffer.
   Mix gently by flicking the tube and incubate for 10 min at room temperature.
   NOTE: Ensure that brains remain submerged and do not adhere to tube walls.

4. Hybridize probes

1. Prepare a 4 nM probe solution by adding 0.4 pmol of each probe set to Probe Hybridization Buffer for a final volume of 100 µL.
   NOTE: Increase probe concentration up to 16 nM if signal intensity is low in initial stains.
2. Remove prehybridization buffer without disturbing the tissue. Add diluted probe solution to each sample.
3. Incubate samples at 37 °C on a nutator for 16–24 h.

5. Amplify probe signal

1. Bring Amplification Buffer to room temperature. Prepare approximately 200 µL of Amplification Buffer per sample.
2. Equilibrate wash buffer. Warm Probe Wash Buffer to 37 °C using a water bath.
   CAUTION: Probe Wash Buffer contains formamide. Handle under a chemical fume hood.
3. Remove probe solution and store at -20 °C for reuse.
   NOTE: Reuse probe solutions until staining intensity decreases.
4. Wash samples four times for 10 min each in preheated Probe Wash Buffer at 37 °C. Wash samples twice for 5 min each in room-temperature 5x SSCT.
5. Add 100 µL of Amplification Buffer without hairpins. Incubate for 30 min at room temperature.
6. Heat a thermocycler to 95 °C. Add 2 µL of each 3 µM hairpin stock to separate PCR tubes. Prepare one tube for each hairpin species.
   NOTE: Increase amplifier concentration if staining produces sparse puncta or a low signal-to-noise ratio.
7. Incubate hairpins at 95 °C for 90 s. Protect tubes from light and incubate at room temperature for at least 30 min.
8. Prepare amplification solution. Combine hairpins in Amplification Buffer to a final volume of 100 µL per sample.
9. Remove Amplification Buffer from samples. Add hairpin-containing Amplification Buffer.
   Incubate overnight at room temperature on a nutator protected from light (wrapped in foil).
10. Remove amplification solution. Wash samples twice for 5 min in 150 µL of 5x SSCT. Wash samples three times for 10 min in 150 µL of 5x SSCT on a nutator. Perform two brief washes in nuclease-free PBS to remove detergent.

6. Mount brains for imaging

1. Adhere an imaging spacer sticker to a glass slide. Press gently with the bottom end of a forceps to remove trapped air bubbles.
2. Bisect the brain through the ventral nerve cord using forceps.
3. Place the brain in the center of the spacer well. Orient the tissue laterally with the optic lobe facing upward.
4. Add sufficient antifade mounting medium (about 25 µL) to fill the well without introducing air bubbles. Distribute mounting medium evenly using forceps.
5. Place a coverslip over the sample and seal with nail polish.

Results

For scientists studying developmental neurobiology, HCR-FISH provides a robust method for visualizing gene expression patterns and determining the spatial positions of developing neurons within the Drosophila optic lobe.

In the Drosophila optic lobe (Figure 2A), neurons within the medulla neuropil arise from neural stem cells (neuroblasts) derived from a spatially patterned neuroepithelium called the Outer Proliferation Center (OPC) (Figure 2B)²⁸. These neuroblasts express sequential transcription factors that generate neuronal diversity over time. Consequently, neurons are arranged in concentric rings according to birth order^{29,30,31,32,33}. Early-born neurons localize closest to the neuropil, whereas later-born neurons occupy progressively more peripheral positions within the developing optic lobe^30,33. Additional optic lobe neurons arise from distinct progenitor domains; lamina neurons originate from the lamina precursor cells (LPCs) at the inner edge of the OPC, whereas many lobula complex neurons arise from the Inner Proliferation Center (IPC) (Figure 2B).

Single-cell transcriptomic approaches have enabled the identification of spatial and temporal transcription factors that regulate neuronal specification within the developing optic lobe^2,33,34. Although these methods provide extensive molecular information, they often lack sufficient spatial resolution to define the position of neuronal birth. HCR-FISH enables direct visualization of transcriptionally defined neuronal populations within intact tissue.

For example, HCR-FISH corroborates previous findings regarding the spatiotemporal origins of Distal medulla (Dm) neurons. The Dm2 neuronal subtype expresses the transcription factor Distal-less (Dll), the cell-adhesion molecule Fas2, and the transcription factor hamlet (ham) (Figure 2C). Because Fas2 protein localizes predominantly to neuronal processes, immunohistochemistry does not clearly identify Dm2 cell bodies³⁵. In contrast, Fas2 transcripts localize within the soma and are readily detected by HCR-FISH. Representative staining demonstrates that Dm2 neurons occupy the posterior edge of the developing optic lobe, consistent with a later-born neuronal identity (Figure 2C). Their distribution along the dorsoventral axis also supports previous single-cell RNA sequencing data, indicating that Dm2 neurons arise from multiple spatial domains within the optic lobe³⁶. In comparison, a ham-GFP reporter line fails to adequately label Dm2 neurons during development (Figure 2C’).

Another medulla neuron subtype, Dm4, arises from stem cells within the ventral neuroepithelium in a domain smaller than previously predicted from transcriptomic datasets alone^36,37. To determine the spatial origin of Dm4 neurons more precisely, HCR-FISH was performed against the Dm4-specific transcription factor abrupt. HCR-FISH detected discrete puncta corresponding to a restricted population of cells within the predicted neurogenic region (Figure 2D). Because abrupt expression is weak during the third larval instar, ab-GFP reporter lines and anti-Abrupt immunostaining fail to clearly label Dm4 neurons (Figure 2D’, 2D’’). HCR-FISH also detected abrupt expression within fenestrated glia located at the lateral edge of the optic lobe (Figure 2E), consistent with previous single-cell RNA sequencing datasets²¹. This expression pattern was not observed using ab-GFP reporters (Figure 2E’).

Figure 2: HCR-FISH labels developing optic lobe neurons in the Drosophila visual system. ISH = in situ hybridization; IHC = immunohistochemistry. (A) Schematic of the Drosophila optic lobe, including the lamina, medulla, lobula, and lobula plate. Photoreceptors R1-R6 project to the lamina, whereas R7-R8 project to the medulla. HCR-FISH was used to label Distal medulla (Dm) neurons Dm2 (purple) and Dm4 (blue). (B) Organization of the third instar larval optic lobe. Neural stem cells (neuroblasts) arise from the Outer Proliferation Center (OPC). Lamina neurons originate from the lamina precursor cells (LPCs) located at the inner edge of the OPC. The medulla is positioned beneath the lamina. Many lobula complex neurons arise from the Inner Proliferation Center (IPC). (C) HCR-FISH against Fas2, Distal-less (Dll), and hamlet (ham) labels Dm2 neurons (arrows). (C’) ham-GFP reporter expression does not accurately label Dm2 neurons in the developing medulla (dotted line indicates medulla neuropil). (D) HCR-FISH against abrupt (ab) labels zfh1-positive Dm4 neurons (arrow), but not CG4328-positive Dm1/Dm12 neurons, consistent with previous single-cell RNA sequencing datasets^21,37. (D’) Anti-Abrupt immunohistochemistry does not clearly label SoxN-positive, Tj-positive Dm4 neurons (arrow). (D’’) ab-GFP reporter expression is weak in the developing medulla, limiting characterization of endogenous abrupt expression patterns (dotted line indicates medulla neuropil). (E) HCR-FISH detects abrupt (ab) expression in fenestrated glia (dotted lines) near domains of brk and dpp expression. (E’) ab-GFP reporter expression does not recapitulate endogenous expression in fenestrated glia (dotted lines). Please click here to view a larger version of this figure.

HCR-FISH also enables characterization of endogenous gene expression patterns when reporter lines fail to accurately recapitulate transcriptional activity. Previous studies demonstrated that BMP/Dpp signaling gradients regulate specification of multiple medulla neuron classes during optic lobe development^37,38. These signaling domains exhibit spatially restricted expression patterns within the optic lobe (Figure 3A). Dpp is expressed at the posterior edge of the optic neuroepithelium, where signaling induces phosphorylation of the transcription factor Mad. Activated pMad promotes expression of optomotor blind (omb) while repressing brinker (brk), generating mutually exclusive expression domains. The BMP type I receptor thickveins (tkv) is expressed within the anterior neuroepithelium and functions together with the spatial transcription factors Vsx, Optix, and Rx to regulate neuronal specification^37,39.

Although expression patterns for several BMP pathway components have been characterized previously, the distribution of additional pathway members within the optic lobe remained unclear. HCR-FISH targeting the BMP type II receptor wishful thinking (wit) demonstrated broad expression within the developing optic neuroepithelium and newborn neurons (Figure 3B-B’). However, a wit-GAL4 reporter line did not accurately reproduce endogenous expression patterns within the brain (Figure 3C’).

The BMP type II receptor punt (put) displayed a related but distinct expression pattern, with stronger expression in neurons of the medulla and lobula neuropils (Figure 3D-D’). Although both wit and put were broadly expressed, their expression domains were not identical (Figure 3D’’), suggesting that these receptors may function within distinct developmental contexts.

Similarly, whereas the canonical Smad Mad is expressed within a restricted signaling domain, the co-Smad Medea exhibited broader expression throughout the optic lobe (Figure 3A, 3E-E’’)³⁷. To compare these expression domains with BMP signaling regions, HCR-FISH was performed in optic lobes expressing a nuclear GFP reporter driven by dpp-GAL4. Endogenous GFP fluorescence remained sufficiently strong after fixation and did not require additional immunostaining (Figure 3B’, 3D’, 3E’).

Collectively, these representative results demonstrate that HCR-FISH provides a reliable and sensitive method for mapping gene expression patterns within the developing Drosophila visual system. The method is particularly valuable when enhancer-GAL4 lines or antibody-based approaches fail to accurately reflect endogenous transcriptional activity.

Figure 3. HCR-FISH reveals BMP pathway gene expression patterns in the developing optic neuroepithelium. (A) BMP signaling components exhibit spatially restricted expression patterns within the developing optic lobe (adapted from Malin et al., 2024)³⁷. Spatial transcription factors Vsx, Optix, and Rx are expressed along the dorsoventral axis³⁹. Dpp = Decapentaplegic; pMad = phosphorylated Mad; Omb = Optomotor blind; Brk = Brinker; Tkv = Thickveins. D = dorsal; V = ventral; A = anterior; P = posterior; L = lateral; M = medial. (B-B’, D-E’’) HCR-FISH of BMP pathway components in optic lobes expressing dpp-GAL4; UAS-nls-GFP (Stinger). Endogenous GFP fluorescence remained detectable after fixation and did not require additional immunostaining. ISH = in situ hybridization; IHC = immunohistochemistry. (B) HCR-FISH against the BMP type II receptor wishful thinking (wit) labels a crescent-shaped domain overlapping the developing optic neuroepithelium and newborn neurons. (B’) wit HCR-FISH labeling in combination with dpp-GAL4; UAS-nls-GFP expression. (C) wit-GAL4; UAS-myr-GFP reporter expression does not accurately recapitulate endogenous wit expression patterns observed by HCR-FISH. (D) HCR-FISH against the BMP type II receptor punt (put) labels a narrower expression domain within the developing optic lobe. (D’) put HCR-FISH labeling in combination with dpp-GAL4; UAS-nls-GFP expression. (D’’) Co-labeling of wit and put demonstrates partially overlapping but distinct expression domains in developing optic lobe neurons. (E) The co-Smad Medea (Med) exhibits an HCR-FISH expression pattern similar to those of wit and put. (E’) Med HCR-FISH labeling in combination with dpp-GAL4; UAS-nls-GFP expression. (E’’) Co-labeling of wit and Med demonstrates broader Med expression in developing neurons. Please click here to view a larger version of this figure.

Discussion

This study describes an adaptation of HCR-FISH for use in the Drosophila melanogaster larval optic lobe. Conventional in situ hybridization approaches often perform poorly in whole-mount larval brain tissue due to limited probe penetration and low signal intensity. The protocol presented here optimizes several critical steps to improve staining quality in the developing optic lobe. Fixation conditions commonly used for Drosophila immunohistochemistry were adapted to preserve tissue morphology while maintaining RNA integrity. Increased probe and amplifier concentrations improved signal intensity and signal-to-noise ratio, particularly for weakly expressed transcripts such as abrupt. In addition, shorter probe lengths improved tissue penetration throughout the approximately 100 µm-thick larval brain, including regions where DAPI staining frequently exhibits limited penetration (Figure 2D).

Several technical considerations are critical for the successful implementation of this protocol. During dissection, optic lobes must remain fully submerged in buffer to prevent tissue dehydration prior to fixation. Because HCR-FISH detects RNA transcripts, all steps should be performed under RNase-free conditions to minimize RNA degradation. Proper hairpin preparation is also essential for successful signal amplification. Hairpins must be denatured and cooled separately before amplification to prevent premature polymerization prior to probe binding.

HCR-FISH provides several advantages over conventional approaches for studying Drosophila neurodevelopment. The method complements immunohistochemistry and GAL4 reporter systems by providing direct spatial information about endogenous transcript localization. Because transcription precedes protein accumulation, RNA-based detection can reveal cell-type-specific markers earlier than antibody staining or reporter expression^33,40,41. This feature is particularly valuable for identifying newly born neurons and characterizing early stages of neuronal specification. Compared with previously published HCR-FISH methods developed for larger insect tissues, the present protocol incorporates shorter wash and hybridization times optimized for the smaller Drosophila larval brain. Use of PCR tubes instead of spot plates also reduces reagent consumption and simplifies handling of small tissue samples.

Despite these advantages, several limitations should be considered. Weakly expressed genes may produce faint hybridization signals, particularly when transcript abundance is low. Signal intensity can often be improved by increasing probe concentration or expanding the number of probes within a probe set. In addition, HCR-FISH may not reliably label structural landmarks required for tissue orientation during imaging. For example, proteins such as N-Cadherin are commonly used to visualize the developing neuropil during immunohistochemistry, whereas their corresponding transcripts exhibit broader, more diffuse localization patterns by FISH. Combining HCR-FISH with HCR-compatible immunohistochemistry (HCR-IHC) can therefore improve anatomical interpretation when additional structural information is required²⁰.

Another limitation is that transcripts expressed within the same cell may localize to distinct subcellular regions. Consequently, overlapping transcript signals do not always precisely define cell identity or cellular boundaries. Although HCR-FISH effectively identifies spatial gene expression domains, additional approaches may be required to confirm specific cell types. Combining HCR-FISH with GFP reporter lines, GFP-targeted probes, HCR-IHC against GFP, or infrared fluorescent nuclear markers can improve positional accuracy and facilitate cell identification⁴².

Overall, HCR-FISH provides a sensitive and versatile method for studying gene expression during neural development in the Drosophila visual system. In addition to mapping neuronal birth positions, future applications may enable analysis of isoform-specific expression patterns, visualization of mRNA splicing dynamics, and imaging of transcriptional regulation upstream of protein translation. When integrated with existing genetic and imaging approaches, HCR-FISH offers a powerful platform for investigating the molecular mechanisms that regulate neuronal specification and circuit assembly in the developing brain.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10% Tween-20	Bio-Rad Laboratories	1662404	NA
20x PBS, pH7.4	Growcells.com	MRGF-6396	2.74M NaCl, 53.7 mM KCl, 20.3 mM Na2HPO4, 29.4 mM KH2PO4
20x SSC	Quality Biological	351-003-131	3M NaCl, 0.3M Sodium Citrate
37 °C incubator	Fisher Scientific (Isotemp)	15-015-2633	NA
37 °C water bath	Fisher Scientific	FSGPD15D	NA
Aluminum foil	Reynolds Wrap/Fisher Brand	15-078-291	NA
Antibody: Mouse-anti-Abrupt	DSHB	Abrupt; RRID: AB_528344	Dilution: 1:20
Antibody: Chicken anti-GFP	Millipore Sigma	Catalog# 06-896; RRID: AB-619712	Dilution: 1:500
Antibody: Rabbit anti-GFP	Life Technologies	#11122; RRID: AB_221569	Dilution: 1:500
Antibody: Rabbit anti-Dll	Genscript/Desplan lab	-	Dilution: 1:200
Antibody: Rat anti-NCadherin	DSHB	DN-Ex #8, RRID: AB_528121	Dilution: 1:50
Antibody: Rabbit anti-SoxN	Genscript/Desplan lab	-	Dilution: 1:200
Antibody: Guinea Pig anti-Traffic jam	Gift from D. Godt	-	Dilution: 1:2,000
Antibody: Donkey anti-Rat 405	Jackson ImmunoResearch	Code # 712-475-150; RRID: AB_2340680	Dilution: 1:100
Antibody: Donkey anti-Chicken 488	Jackson ImmunoResearch	Code #703-545-155; RRID: AB_2340375	Dilution: 1:200
Antibody: Donkey anti-Mouse 488	Jackson ImmunoResearch	Code #715-545-151; RRID: AB_2341099	Dilution: 1:200
Antibody: Donkey anti-Rabbit 488	Jackson ImmunoResearch	Code #711-545-152; RRID: AB_2313584	Dilution: 1:200
Antibody: Donkey anti-Rabbit A555	Thermo Scientific	Catalog #A-31572; RRID: AB_162543	Dilution: 1:200
Antibody: Donkey anti-Rat Cy3	Jackson ImmunoResearch	Code #712-165-153; RRID: AB_2340667	Dilution: 1:200
Antibody: Donkey anti-Guinea Pig 647	Jackson ImmunoResearch	Code #706-605-148; RRID: AB_2340476	Dilution: 1:200
Antibody: Donkey anti-Rat 647	Jackson ImmunoResearch	Code #712-605-153; RRID: AB_2340694	Dilution: 1:200
Antifade mountant	Thermo Fisher Scientific	S36940 (SlowFade Gold Antifade Mountant)	Other slowfades will work as well, including ones with DAPI (S36936)
Confocal Microscope	Zeiss	LSM-880	NA
Cover slips (22 x 30)	VWR	16004-332	NA
Fly: D. melanogaster: ab-GFP-FLAG	BDSC	38626; RRID: BDSC_38626	NA
Fly: D. melanogaster: Canton S	BDSC	64349; RRID: BDSC_64349	NA
Fly: D. melanogaster: dpp-Gal4	BDSC	1553; RRID: BDSC_1553	NA
Fly: D. melanogaster: ham-GFP.FPTB	BDSC	83660; RRID: BDSC_83660	NA
Fly: D. melanogaster: yw; 10xUAS-myr-GFP;	BDSC	32198; RRID: BDSC_32198	NA
Fly: D. melanogaster: UAS-Stinger	BDSC	84277; RRID: BDSC_84277	NA
Fly: D. melanogaster:  wit-KO-Gal4	BDSC	600136; RRID: BDSC_600136	NA
Forceps	Dumont	#5 or #55s	NA
Glass slides	VWR	16005-106	NA
Glycine	Millipore Sigma	G7126-10MG	NA
HCR Amplification buffer	Molecular Instruments	HCR Gold RNA-FISH Kit	NA
HCR-Gold Amplifiers	Molecular Instruments	HCR Gold RNA-FISH Kit	NA
HCR HiFi Probes	Molecular Instruments	HCR Gold RNA-FISH Kit	NA
HCR Probe Hybridization Buffer	Molecular Instruments	HCR Gold RNA-FISH Kit	Contains formamide
HCR Probe Wash Buffer	Molecular Instruments	HCR Gold RNA-FISH Kit	Contains formamide
Nail polish (clear)	Electron Microscopy Sciences	72180	NA
Nuclease-free water (not DEPC-treated)	Thermo Fisher Scientific	AM9937	NA
Nutating mixer	Corning/Fisher Scientific	10-320-100	NA
Paraformaldehyde, 16% (w/v)	Electron Microscopy Sciences	15710	Toxic
PCR Machine/Thermal Cycler	Bio-Rad	1851148 (C1000 Touch Thermal Cycler)	NA
PCR pull-apart tube strips with dome cap strips (0.2 mL)	USA Scientific	#1402-2400	NA
Petri dishes (150 x 15 mm; for making dissecting dishes)	Fisher Scientific	FB0875714	NA
RNase ZAP	ThermoFisher Scientific	AM9780	NA
Slide spacer stickers	SUNJin	IS016 (iSpacer stickers (0.2 mm deep))	NA
Sylgard elastomer kit (for making dissecting dishes)	Fisher Scientific	NC9285739	NA
Triton X-100	Merck Millipore Sigma	X100	NA