Precise Visualization of Insulin Receptors A and B in Murine Brain with an RNA In Situ Hybridization Assay

Summary

We established a Duplex in situ hybridization assay to map insulin receptor isoforms IR-A and IR-B in the murine choroid plexus, overcoming limitations of traditional methods. This high-resolution approach reveals isoform-specific expression, offering new insights into brain insulin signaling and potential isoform-targeted therapies for neurological disorders.

Abstract

Insulin receptors (IRs) are widely expressed across all tissues and all cell types. It exists in two functionally distinct isoforms -- IR-A and IR-B -- differing by 12 amino acids due to alternative exon 11 splicing. These isoforms exhibit tissue- and cell-specific expression patterns, with their relative abundance varying throughout development, aging, and disease. Due to their high sequence similarity, distinguishing between IR-A and IR-B using traditional immunological methods has been challenging. Here, we describe a novel Duplex in situ hybridization (ISH) assay that enables the visualization of IR-A and IR-B expression in tissue. Using this approach, we demonstrate for the first time that while both isoforms are expressed in the murine choroid plexus, IR-A is predominant. This method provides a powerful tool for investigating the spatial distribution of insulin receptor isoforms in situ across different tissues. While real-time qPCR can detect IR-A and IR-B transcripts in monocellular in vitro cultures, it lacks the capacity for spatial localization within complex tissues, highlighting the need for and utility of the Duplex ISH technique.

Introduction

The insulin receptor (IR) is a single-pass transmembrane enzymatic receptor that mediates the diverse physiological effects of insulin. It functions as a dimeric protein, with each monomer composed of two peptide chains linked by a disulfide bond: The extracellular α-subunit, which contains the ligand-binding domain, and the transmembrane β-subunit, whose intracellular sequence possesses intrinsic tyrosine kinase activity¹. Through alternative splicing of exon 11, transcription of the IR gene gives rise to two IR mRNA variants. IR-A lacks exon 11, while IR-B retains it. Consequently, IR-A and IR-B differ by a 12-amino acid sequence near the C-terminus of the α-subunit, resulting in distinct binding of growth factors besides mature insulin, and varying downstream signaling properties. While both variants exhibit similar affinity for insulin, IR-A has a higher affinity for insulin-like growth factor-II (IGF-II) and proinsulin compared to IR-B. The expression patterns of IR-A and IR-B vary depending on tissue and cell type, and disease state².

Their differential expression in tissues and disease underscores their unique contribution to physiology and pathology. The choroid plexus (CP), a specialized structure that produces cerebrospinal fluid (CSF) and forms part of the blood-CSF barrier, plays a critical role in nutrient transport, waste clearance, and neuroprotection, especially during inflammation. Both IR isoforms are expressed in the CP, where they likely regulate CSF volume and composition. IR-A, through its high-affinity binding by IGF-II, likely promotes cellular proliferation and tissue repair in the CP epithelium. IR-B, on the other hand, is implicated in insulin transport across the blood-CSF barrier, supporting insulin availability in the brain for astrocytes, neurons, glia, and glucose homeostasis^3,4.

Pre-diabetic IR levels have been associated with elevated CSF tau in asymptomatic individuals at genetic risk for Alzheimer's disease (AD)⁵, and alterations in the IR-A/IR-B ratio have been linked to insulin resistance, aging, and increased proliferative activity-factors that may contribute to cancer and other pathologies⁶. Identifying isoform-specific expression patterns of IR-A and IR-B represents a critical step toward developing novel therapeutic strategies for complex central nervous system disorders. However, current approaches, such as immunostaining and qPCR, are limited: Not all proteins can be reliably immunolabeled due to a lack of validated antibodies, and while qPCR is highly specific, it lacks spatial resolution.

To overcome these limitations, we utilized Duplex in situ hybridization (ISH), an advanced RNA detection technology based on the RNAscope platform BaseScope. This assay employs a unique double-Z probe design that requires tandem binding to the target RNA, enabling high specificity and sensitivity, even for low-abundance transcripts. The fluorescent or chromogenic signal is amplified through multiple probe-binding events, enhancing detection and reducing background⁷. Unlike standard RNAscope, this assay is optimized to detect short RNA sequences (50-300 bases), making it particularly suitable for identifying alternative splicing events, single-nucleotide variants, and rare transcripts in complex tissues like the brain⁸.

This assay visualizes targets simultaneously as distinct green (C1) and red (C2) chromogenic signals under brightfield. It ensures success using tissues fixed in 4% paraformaldehyde (PFA) or 10% neutral buffered formalin (NBF) and sectioned at 5 µm-15 µm (frozen/FFPE), containing low-to-high abundance RNA. Due to sensitivity to RNA degradation, over-fixation, and probe errors, we need to optimize pretreatment, probe design, and controls to maximize specificity and minimize background. Here, we applied this Duplex ISH assay to characterize the expression patterns of IR-A and IR-B in the murine CP, offering new insights into the spatial biology of IR isoforms and their possible roles in brain health and disease.

Protocol

All animal procedures were conducted in compliance with protocols approved by the National Institute on Aging (NIA), which is fully accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC). Protocols were reviewed and approved by the Animal Care and Use Committee of the NIA Intramural Research Program (IRP/NIA). Mice were housed in a temperature-controlled facility under a 12 h light/dark cycle, with ad libitum access to water and standard chow unless otherwise specified. C57BL/6J male mice were obtained from the Jackson Laboratory and used for experiments at 3–4 months of age.

1. Fixed frozen sample preparation

1. Perfuse the mouse with freshly prepared 4% PFA in 1x PBS and place the whole brain in freshly prepared 4% PFA for 17–24 h at 4 °C.  
   NOTE: Tissue placed in 4% PFA (10 mL for each brain) for no more than 24 h.
2. Immerse the tissue in 10% (10 mL) sucrose in 1x PBS at 4 °C until the tissue sinks to the bottom of the container. Then, sequentially transfer the tissue to 20% sucrose and finally to 30% sucrose, allowing it to sink at each step.
   NOTE: The time needed for the tissue to sink varies with tissue type and size. For brain tissue, it takes approximately 18 h.
3. Freeze the tissue in OCT embedding media and store tissue blocks in an airtight container at -80 °C.
4. Coronally section formaldehyde-fixed frozen brain tissues into 10 µm slices and store at −80 °C until use.
   NOTE: Mount the sections on positively charged microscope slides; other slide types may result in tissue loss. If slides are not used immediately, store them at -80 °C for up to 3 months.

2. Section pretreatment prior to duplex detection assay

1. On the day of the assay, wash the slides in 1x PBS for 5 min, gently moving the slide rack up and down to remove any residual OCT.
2. Bake the slides at 60 °C for 30 min.
3. Post-fix the slides in prechilled 4% PFA in 1x PBS for 15 min at 4 °C.
4. Transfer the slides sequentially through an EtOH series: 50% EtOH for 5 min at RT, 70% EtOH for 5 min at RT, 100% EtOH for 5 min at RT, 100% EtOH again for 5 min at RT.
5. Air-dry slides for 5 min at RT.
6. Apply 2–4 drops of hydrogen peroxide to each section and incubate for 10 min at RT. Rinse once with distilled water.
7. Prepare 700 mL of fresh 1x Target Retrieval solution in a beaker. Cover with foil and bring to a consistent boil (99–100 °C).
   NOTE: Do not boil for more than 30 min before use. Meanwhile, preheat the oven (see the Table of Materials) to 40 °C.
8. Carefully immerse the slide rack into the boiling Target Retrieval solution and incubate for 5 min. Maintain the temperature between 98–102 °C and monitor closely.
   NOTE: Boiling time may need to be adjusted (5–15 min) depending on the tissue type.
9. Immediately transfer the hot slide rack into a dish with distilled water, moving the rack up and down 3–5x. Repeat with fresh distilled water.
10. Rinse slides in fresh 100% EtOH, moving them up and down 3–5x. Air-dry slides.
11. Use a hydrophobic barrier pen to draw around each tissue section 2–3x. Allow the barrier to dry completely at RT.
12. Place the slides in the Batch Slide Tray. Add Protease (see the Table of Materials) to each section, ensuring the tissue is fully covered. Incubate in the oven at 40 °C for 15 min.
    NOTE: Protease incubation time may require adjustment depending on tissue type and target gene.
13. Remove the slide tray from the oven and place it into distilled water. Gently wash the slides by moving them up and down for 2 min, repeating this step 2x with fresh distilled water.

3. Duplex detection assay

1. Probes
   1. Prepare two additional sections for positive and negative control probes.
   2. Select control probes with the same number of ZZ pairs as the target probe. For 1 ZZ pair, use a Positive control and a Negative control (see the Table of Materials).
   3. Prior to use, equilibrate probes at 40 °C for 10 min and bring AMP1-12 reagents to RT.
2. Probe Hybridization
   1. Briefly spin down the C2 probe and mix at 1:50 ratio with the C1 probe. Add enough probe (100–200 µL) mix to fully cover each section and incubate the slides in the oven at 40 °C for 2 h. Wash the slides for 2 x 2 min in 1x Wash buffer at RT.
   2. Base Scope Duplex AMP1 Application
      1. Remove excess washing buffer from slides and add AMP1 (100–200 µL) to entirely cover each section. Return to the oven and incubate at 40 °C for 30 min.
      2. Wash 2 x 2 min in 1x Wash Buffer.
   3. Base Scope Duplex AMP2 Application
      1. Remove excess washing buffer from slides and add AMP2 (100–200 µL) to entirely cover each section. Return to the oven and incubate at 40 °C for 30 min.
      2. Wash 2 x 2 min in 1x Wash Buffer.
   4. Base Scope Duplex AMP3 Application
      1. Remove excess washing buffer from slides and add AMP3 (100–200 µL) to entirely cover each section. Return to the oven and incubate at 40 °C for 15 min.
      2. Wash 2 x 2 min in 1x Wash Buffer.
         ​NOTE: Do not exceed 15 min of incubation.
   5. Base Scope Duplex AMP4 Application
      1. Remove excess washing buffer from slides and add AMP4 (100–200 µL) to entirely cover each section. Return to the oven and incubate at 40 °C for 15 min.
      2. Wash 2 x 2 min in 1x Wash Buffer.
   6. Base Scope Duplex AMP5 Application
      1. Remove excess washing buffer from slides and add AMP5 (100–200 µL) to entirely cover each section. Return to the oven and incubate at 40 °C for 30 min.
      2. Wash 2 x 2 min in 1x Wash Buffer.
   7. Base Scope Duplex AMP6 Application
      1. Remove excess washing buffer from slides and add AMP6 (100–200 µL) to entirely cover each section. Return to the oven and incubate at 40 °C for 15 min.
      2. Wash 2 x 2 min in 1x Wash Buffer.
         ​NOTE: As for AMP3, do not exceed 15 min of incubation.
   8. Base Scope Duplex AMP7 Application
      1. Remove excess washing buffer from slides and add AMP7 (100–200 µL) to entirely cover each section and incubate at room temperature for 30 min.
         1. Wash 2 x 2 min in 1x Wash Buffer.
            ​NOTE: Incubate with AMP7 at  room temperature (RT) for 30 min.
   9. Base Scope Duplex AMP8 Application
      1. Remove excess washing buffer from slides and add AMP8 (100–200 µL) to entirely cover each section and incubate at room temperature for 15 min.
      2. Wash 2 x 2 min in 1x Wash Buffer.
         ​NOTE: Incubate with AMP8 incubation for 15 min at RT.
   10. Red Signal Detection (C2)
       1. Briefly spin down the Fast Red-B and mix with Fast Red-A in a 1:60 ratio.
       2. Apply to each section (100–200 µL), cover the tray, and incubate for 10 min at RT in the dark.
       3. Wash 2x in 1x Wash Buffer.
          ​NOTE: Use mixed Fast Red solution within 5 min. Protect from light (sunlight or UV).
   11. Base Scope Duplex AMP9 Application
       1. Remove excess washing buffer from slides and add AMP9 (100–200 µL) to entirely cover each section. Return to the oven and incubate at 40 °C for 15 min.
       2. Wash 2 x 2 min in 1x Wash Buffer.
   12. Base Scope Duplex AMP10 Application
       1. Remove excess washing buffer from slides and add AMP10 (100–200 µL) to entirely cover each section. Return to the oven and incubate at 40 °C for 15 min.
       2. Wash 2 x 2 min in 1x Wash Buffer.
   13. Base Scope Duplex AMP11 Application
       1. Remove excess washing buffer from slides and add AMP11 (100–200 µL) to entirely cover each section. Incubate at RT for 30 min.
       2. Wash 2 x 2 min in 1x Wash Buffer.
          ​NOTE: Incubate with AMP11 at RT for 30 min.
   14. Base Scope Duplex AMP12 Application
       1. Remove excess washing buffer from slides and add AMP12 (100–200 µL) to entirely cover each section. Incubate at RT for 15 min.
       2. Wash 2 x 2 min in 1x Wash Buffer.
          ​NOTE: Do not exceed 15 min of incubation.
   15. Green Signal Detection (C1)
       1. Briefly spin down Fast Green-B tube and mix with Fast Green-A in a 1:50 ratio.
       2. Apply the GREEN solution to each section (100–200 µL), cover the tray, and incubate for 10 min at RT.
       3. Wash for 5 min in 1x Wash Buffer and briefly rinse in distilled water.
          NOTE: Use mixed Fast Green solution within 5 min. Protect from light. Green B to A ratio is 1:50. Both RED and GREEN substrates are alcohol-sensitive. Do not use any reagents containing alcohol.

4. Counterstain

1. Immerse slides in 50% Hematoxylin staining solution for 1 min at RT until sections appear purple.
2. Rinse in tap water 3–5x by moving up and down. Repeat with fresh tap water until the background clears but sections remain purple.
3. Dip slides in 0.02% Ammonia water up and down 3x until the sections turn blue.
4. Rinse again in tap water 3–5x.

5. Mounting

1. Completely dry slides in a 60 °C dry oven for ~15 min.
2. Dip slides in fresh xylene.
   NOTE: As the mounting medium is quite viscous, a brief dip in xylene prior to mounting facilitates smoother spreading of the medium across the tissue section and reduces the formation of air bubbles under the coverslip.
3. Apply mounting medium (see the Table of Materials) on the slide and coverslip, avoiding bubbles.
4. Air dry slides for at least 5 min.

6. Evaluate the samples

1. Image the sections under a standard bright field microscope at 20x or 40x magnification. (Figure 1).
   NOTE: Positive controls should show punctuate dots in cells. Negative controls are acceptable if there is no more than 1 dot per 20 cells at 20x magnification.

7. Scoring (optional)

1. Use image analysis software (e.g., Halo, or QuPath) to quantify the RNA dots per cell.

Results

The insulin receptor (IR) is a transmembrane tyrosine kinase receptor composed of two extracellular α-subunits and two transmembrane β-subunits, forming a functional heterotetramer. The α-subunits mediate insulin binding, while the β-subunits possess intrinsic tyrosine kinase activity that initiates downstream signaling. IR mRNA alternative splicing of exon 11 generates two isoforms of the IR, IR-A and IR-B. As such, IR-B includes a 12-amino-acid segment encoded by exon 11 within the C-te...

Discussion

Within the brain, the expression of insulin receptors (IR) is heterogeneous, with high densities observed in regions such as the olfactory bulb, hypothalamus, hippocampus, and choroid plexus¹⁰. Comprehensive characterization of the variant expression patterns in specific brain regions and cell types would provide crucial information, enabling us to study and understand the specific roles of insulin and IGF-II signaling in synaptic plasticity, learning, and memory¹². Additio...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ammonium Water	Electron Microscope Science	26754-4A
BaseScope Dulplex Detection Reagent Kit	ACD Bio	323810
BaseScope Dulplex Duplex Positive Control Probe- Mouse(Mm)-C1-Ppib-1ZZ/C2-Polr2a-1ZZ	ACD Bio	700121
BaseScope Dulplex Duplex Positive Control Probe-C1-DapB-1ZZ/C2-DapB-1ZZ	ACD Bio	700141
Gill's Hematoxylin I	Sigma-Aldrich	GHS132-1L
Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488	Invitrogen	A32731
HybEZ OVEN, Tray, Rack and Wash Tray	ACD Bio	321721, 310012, 321716, 321717
ImmEdge Hydrophobic Barrier Pen	Vector Laboratory	H-4000
INSR isoform Aand B probes: BA-Mm-Insr-tv1-E10E11, BA-Mm-Insr-tv2-E11E12-C2	ACD Bio	717411, 717421-C2
Insulin Receptor antibody	Cell Signaling	23413
RNAscope 10x Target Retrival	ACD Bio	322000
RNAscope Hydrogen Peroxide, Protease Plus	ACD Bio	322381
Saline-Sodium Citrate (SSC) 20x	Fisher Scientific	BP1325-1
SuperFrost Plus slides	Fisher Scientific	12-550-18
VectaMount Permanent Mounting Medium	Vector Laboratory	H-5000