Analyzing Photoreceptor Degeneration and Preservation in a Retinitis Pigmentosa Murine Model

Summary

This protocol outlines optimized techniques for analyzing photoreceptor degeneration and preservation in a murine model of retinitis pigmentosa, including intravitreal drug administration, cryostat sectioning, and immunofluorescence. The method ensures retinal integrity and provides quantitative data on the effectiveness of therapeutic interventions.

Abstract

Retinopathies affect hundreds of millions of people worldwide and are significant causes of visual impairment and blindness. Among these diseases, retinitis pigmentosa (RP) is a hereditary ocular disorder characterized by progressive retinal degeneration, affecting approximately 1.5 to 2 million people worldwide. Proof of concept studies in animal models of hereditary retinal dystrophies form an important basis of evidence for studies in pathophysiological mechanisms of the disease, treatment effectiveness and safety. Intravitreal injection in mice and cryostat sectioning are widely utilized techniques for investigating drug effects in RP models, as they enable a precise delivery of substances into vitreous humor and preparation of high-quality histological samples for detailed analysis of retinal alterations. Also, cell counting through immunofluorescence using antibodies against recoverin, a calcium-dependent protein expressed in retinal photoreceptors, has been recently employed to study cellular changes associated with retinal degeneration in RP models, because this approach facilitates evaluation of photoreceptor loss and potential cellular preservation following therapeutic interventions. The intravitreal injection has been optimized to ensure ocular integrity and reliable results, utilizing a fine needle to administer the solution into the vitreous chamber. After a predefined period to assess drug effects, histological processing is performed, with samples sectioned at 10 μm thickness using a cryostat, followed by immunofluorescence labeling with recoverin antibodies. Finally, cell counting is performed to assess whether the treatment with drugs exerted a protective effect, as evidenced by preservation of photoreceptors. Although these techniques have been extensively applied to generate consistent data in retinopathy research, challenges persist in achieving precise tissue handling and processing quality. This protocol provides a standardized approach to optimize each step, from intravitreal injection to the preparation of histological sections, ensuring reproducible and high-quality results.

Introduction

Vision is one of the most complex senses of the sensory system, essential for capturing environmental visual stimuli and translating them into electrochemical signals, which are processed in the brain to form images. Various biological factors, such as aging, genetic defects, and diseases, can compromise vision, significantly impacting quality of life and daily activities.

The human eye consists of three primary chambers. The outer layer, formed by the cornea and sclera, provides structural support to the eyeball. The intermediate layer includes the iris, ciliary body, and choroid, with the latter responsible for supplying blood to the photoreceptors. Lastly, the inner layer comprises the retina, a structure responsible for processing light stimuli and converting light into electrical signals sent to the brain for image formation^1,2.

The neural retina is composed of several cellular layers, including specialized cells such as photoreceptors, bipolar cells, horizontal cells, amacrine cells, and ganglion cells. Additionally, the retina contains glial cells such as microglia, astrocytes, and Müller cells, as well as pigmented epithelial cells. The latter, located in the non-neural portion of the retina, plays a critical role in maintaining structural integrity, protection, and support to photoreceptors^3,4. The neural portion houses sensory and neuronal cells directly involved in light capture and processing^5,6,7.

Being essential for vision, the retina is often affected by pathologies that can lead to blindness. Among these conditions, retinitis pigmentosa (RP) stands out as one of the main hereditary retinopathies, with more than 80 genes associated with its etiology^8,9. RP is a heterogeneous disease, predominantly inherited in an autosomal dominant or recessive manner, but it may also occur as X-linked or due to spontaneous mutations¹⁰.

With a global prevalence estimated between 1 in 3,000 and 1 in 7,000 individuals, depending on the population and geographic region^11,12, RP usually manifests during adolescence or early adulthood, with initial symptoms such as decreased night vision and peripheral visual field loss. As the disease progresses, central vision becomes compromised, potentially leading to total blindness^9,13.

Although there is no cure for RP yet, various therapies in development have shown potential to slow or reverse disease progression. Among these approaches are genetic, cellular, and pharmacological therapies, as well as electronic devices. To advance the understanding of RP and evaluate new interventions, murine models of retinal degeneration have been widely used. These models, often referred to as rd (retinal degeneration), are valuable tools for studying the molecular basis and therapies for RP. Among the various models used in the study of retinitis pigmentosa, the Pde6β^rd10/rd10 (rd10) mouse strain presents a missense mutation in the gene encoding the rod-specific β subunit of phosphodiesterase¹⁴, exhibiting a slow degeneration of photoreceptors similar to what occurs in humans¹⁵.

Intravitreal drug administration has been widely used in various studies^16,17,18. This protocol is suitable for researchers studying drug effects on the retina, particularly regarding photoreceptor cell survival. In this way, the primary objective of this protocol is to optimize drug delivery via intravitreal injection in a murine model of retinitis pigmentosa (RP) and assess photoreceptor survival using immunofluorescence for recoverin, a photoreceptor cell marker. Intravitreal injection enables localized drug administration, minimizing systemic side effects, increasing retinal bioavailability, and reducing adverse effects in other organs. On the other hand, immunofluorescence for recoverin allows a specific analysis of photoreceptor survival, providing an effective tool to evaluate the efficacy of this therapeutic strategy. However, their applicability depends on factors such as animal model selection, injection precision, and treatment duration, which should be considered when adapting this method to other experimental settings.

Protocol

The project and all the protocols carried out within it follow the animal care guidelines of the National Council for the Control of Animal Experimentation and were approved by the Ethics Committee on Animal Use (CEUA - UFF) under the number 1464280219.

1. Animals

1. Use the rd10 murine strain, which undergoes photoreceptor degeneration, and the C57 Black/6 strain (background of rd10). Maintain these strains under a 12 h/12 h light-dark cycle, with ad libitum access to water and food.

2. Anesthesia

1. Perform an intraperitoneal injection with an anesthetic prepared at a dose of 80 mg/kg of ketamine and 8 mg/kg of xylazine, diluted in distilled water. Ketamine acts as an N-methyl-D-aspartate receptor (NMDA) receptor inhibitor, providing analgesic effects, while xylazine functions as an anesthetic sedative.
2. For the intraperitoneal injection, calculate the volume to be injected as 10 µL x Weight (g).
3. Restrain the mouse to ensure clear access to the abdomen. Clean the lower right quadrant of the abdominal cavity with 70% alcohol, where the needle should be inserted with the bevel facing up.
4. To confirm the absence of a pain reflex, gently pinch the animal's tail or paw. If there is no reaction, this indicates the animal is properly anesthetized.

3. Intravitreal injection

1. With the animals anesthetized, expose the eyes and, using a binocular stereoscopic microscope with a magnification of 10x, identify the limbus area, a gray band between the cornea and sclera, where the injection will be performed.
2. To perform the injection, assistance from another person is needed. Under a binocular stereoscopic microscope, one person has to hold the animal and insert the needle, while the assistant presses the syringe plunger to introduce the substance into the eye.
3. First, use an ultra-fine syringe with a fixed needle (31 G x 6 mm) to make a puncture in the limbus region, being careful not to insert the needle too deeply; inserting only half of the bevel is sufficient.
4. Then, insert the handheld pre-sterilized Micro Syringe (gauge 26 S without bevel), containing 1 µL of the liquid/drug of interest, into the puncture made by the insulin syringe.
5. Have the assistant press the syringe plunger slowly, ensuring that the entire volume is administered over approximately 30 s.
6. Repeat the process for the other eye of the same animal and record the time.
7. If there is more than one experimental group, mark the animal's ear to ensure proper identification of each group.
8. Use 1 drop of 0.01 M PBS solution in each eye of the animal to prevent drying.
9. After these procedures, put the animals on a heating pad, with the temperature set to 37 °C, until they wake up. It can take 1-2 h. Monitor the animals continuously while anesthetized, and once awake, return them to their cages until the time of analysis.
10. Carry out an initial training session using a visible dye, such as trypan blue, to help operators refine their intravitreal injection technique in mice.
    NOTE: The dye facilitates the visualization of the needle path, allowing precise adjustments in depth and angle of the injection, reducing the risk of damage to ocular structure and increasing the safety of the procedure.

4. Euthanasia and eye preparation

1. After a specific duration of treatment following the injection, euthanize the animals via inhalation of a 5% isoflurane overdose. Continue the exposure until at least 1 min after the cessation of breathing. To ensure death, hold the animal by the back of the neck and apply firm traction on the tail until cervical dislocation is achieved.
2. Before removing the eyes, mark the temporal pole. Carefully apply the marker or hot needle/pin to the temporal region of the sclera, ensuring that the marking is visible and precise. This step will serve as a reference point for orientation during the sectioning process.
3. Next, expose the eyes and, with a sterile scalpel and iridectomy scissors, cut the surrounding tissue to separate connective and muscle tissues and expose the eyeball. In mice, dissect the eye muscles carefully, as this can be challenging. To facilitate the procedure, make a lateral incision with a sterile scalpel at both corners of the eye skin.
4. Using a medium-tip curved forceps, gently remove the eyes from the eye socket by holding them by the optic nerve and immediately fix the tissue for 5 min in a 4% paraformaldehyde (PFA) solution at room temperature (RT).
5. After fixation in PFA solution, transfer the eyes to a glass plate, using medium tip curved forceps. This plate functions as a dissection platform and ensure it contains a phosphate buffer at a concentration of 0.1 M pH 7.4.
6. Next, place the plate under a binocular stereoscopic microscope with a magnification of 10x, and make a small incision just above the limbus (corneal border) using a scalpel. Subsequently, perform a circumferential cut following the limbus with microiridectomy scissors to separate the cornea.
7. Remove the cornea, along with the iris and the lens, using Dumont #5 forceps. Finally, fix the eyes again in the solution of 4% PFA for 55 min. After this, wash the eyes three times with phosphate buffer 0.1 M, pH 7.4.

5. Cryopreservation and freezing

NOTE: Retinal tissue cryostat sectioning was performed as described previously with some adjustments¹⁹.

1. Cryopreserve the eyes with a sucrose gradient of 10%, 20%, and 30%, diluted in 0.1 M phosphate buffer, pH 7.4. Submerge the eyes sequentially in the sucrose solutions, starting with the lowest concentration (10%) and gradually increasing to the highest (30%). Each step lasts for the necessary time until the eyes settle at the bottom of the container, indicating that the next concentration can be used. In the final concentration, store the eyes at 4 °C for approximately 12 h.
2. The following day, remove the excess 30% sucrose and place the eyes in a 2 cm diameter round aluminum container, arranged according to the experimental groups.
   NOTE: Make an aluminum foil container in the appropriate size according to the number of experimental groups.
3. Under a binocular stereoscopic microscope with a magnification of 10x, remove the excess sucrose from inside and around the eyeball using a pipette or filter paper, taking the necessary care not to damage the tissue structure.
4. Embed the eyes in optimal cutting temperature (OCT) freezing medium, taking care to avoid the formation of bubbles inside or around the eyes, and leave them for 1 h at RT.
5. After 1 h in OCT, position the eyes with the previously marked reference point facing up. Place the aluminum foil container on a suitable aluminum plate inside a small styrofoam box for freezing.
6. Carefully pour liquid nitrogen onto the aluminum plate, around the foil container. Ensure that the nitrogen spreads evenly across the surface. This promotes uniform tissue freezing. Make sure the liquid nitrogen does not come into direct contact with the eyes inside the foil container. Then, proceed with sectioning the eyes using a cryostat.

6. Slide preparation

1. Spread poly-L-lysine (200 μg/mL) on the frosted-edge glass slides.
2. Wait for 12 h for the slides to dry and be ready for use.

7. Cryostat sectioning

1. On a cryostat, position the frozen block on the platform.
2. Turn the handwheel to make sections with a thickness of 10 μm.
3. Adjust the position of the block to ensure that all eyes are equally present in the cut.
4. Continue cutting until the optic nerve is identified, using a bright-field microscope.
5. Collect the sections on the poly-L-lysine pre-treated slides.
6. Store the slides at -20 °C until analysis.

8. Immunofluorescence

NOTE: Immunofluorescence was performed as described previously with some adjustments^19,20.

1. Incubate the slides obtained from the section in the cryostat with 100 μL of 0.5% Triton X-100 in 0.01 M PBS for 15 min at RT to permeabilize the cell plasma membrane.
2. Wash the slides twice with 0.01 M PBS for 5 min each at RT to remove excess Triton X-100.
3. Incubate with 100 μL 1% bovine serum albumin (BSA) in 0.01 M PBS for 30 min at RT to block non-specific antibody binding sites.
4. After blocking, incubate the sections with 100 μL of anti-recoverin at a concentration of 1:1500 diluted in BSA 1% to label photoreceptor cells, overnight at 4 °C (refrigerator).
5. On the following day, incubate the sections with 100 μL of anti-rabbit polyclonal secondary antibody 488 or anti-rabbit polyclonal secondary antibody 568 1:500 diluted in BSA 1% for 2 h at RT.
6. Wash the slides twice with 0.01 M PBS for 5 min each at RT.
7. Incubate the sections with 100 μL of DAPI 1 mg/mL (4',6-diamidino-2-phenylindole) for 2-3 min at RT.
8. Wash the slide once with 0.01 M PBS for 5 min at RT.
9. Finally, treat the sections with a mounting medium containing 2.5 g of n-propyl gallate in 40 mL of glycerol in 0.1 M phosphate buffer pH 7.4. They will act as an anti-fade reagent and a mounting medium, preserving the samples. Place a coverslip (24 mm 50 mm) on top of the slides, then seal the sections with nail polish. After that, proceed with viewing the samples under the microscope.

9. Fluorescence microscope photomicrography

1. To acquire images of the cells labeled using the immunohistochemistry technique, use a fluorescence optical microscope equipped with blue, green, and red filters. Additionally, attach a camera for slide photomicrography. To obtain the photomicrographs, use a computer with the LAS V3.7 (Leica) software (imaging software), which must be open for simultaneous visualization with the microscope.
2. Place the slide on the microscope stage. Initially, use the lowest magnification objective and the blue filter.
3. Adjust the focus using the coarse and fine focus knobs until the retina with the labeled cells in blue (cell nuclei) is clearly visualized.
4. Select the desired area of the retina for imaging. Gradually increase the magnification up to 40x objective, adjusting the focus as needed.
5. After adjusting the focus, pull the bypass pin to transfer the microscope image to the imaging software. Select the desired filters (A4 (blue), L5 (green), and TX2 (red)). Adjust the following parameters: Exposure, Gain, and Gamma, to ensure the image is clear and as close as possible to what is observed under the microscope.
6. At 400× magnification, capture four fields with an area of 113 × 113 μm, following an orientation from the periphery to the center of the retina. Ensure two of these fields represent the peripheral region, and two represent the central region of the retina.
7. To obtain peripheral field photomicrographs, capture images at approximately 50 μm from the edges of the eye. For central field photomicrographs, select adjacent areas located 50 μm from the optic nerve and acquire the images. Use both right and left eyes for quantifications, resulting in a total of eight analyzed fields per treatment.
8. After completing all the photomicrographs, proceed with the manual quantification of the immunolabeled cells.

10. Cell quantification

1. Open the ImageJ v.1.54g program. Within the program, click on File, then Open, and select the image saved on the computer.
2. With the image already open, click on Multi-point. Use this tool to quantify the cells by left-clicking on each cell to be counted.
3. Quantify the colocalized cells, i.e., positive for recoverin and DAPI, in the outer nuclear layer (ONL).
4. When the count is complete, double-click with the left mouse button to see the total number of cells that were marked.
5. After determining the number of recoverin-positive cells, enter the number of cells in the Excel quantification table and move on to the next photomicrograph.
6. Repeat all steps for the 8 fields corresponding to each animal.
7. Calculate the average of these eight fields, corresponding to a single experimental point for the entire retina (n= 1). For the peripheral retina graph, use the average of the peripheral fields, totaling four fields: two from the left eye and two from the right eye. This average corresponds to one experimental point for the peripheral retina (n= 1). Create the center of the retina graph in the same manner as the peripheral graph, changing only the region.

Results

The immunofluorescence technique for labeling photoreceptors, recoverin-positive cells, allowed us to observe the degeneration pattern of photoreceptors in rd10 and C57Bl6 animals, with the latter considered healthy (Figure 1). As can be seen, labeling for recoverin-positive cells is exclusive to the outer nuclear layer (ONL) where photoreceptors are found, including labeling of segments internal to these cells, both in rd10 and wild-type animals. As developm...

Discussion

The retina is a delicate tissue that is difficult to access; therefore, the technique to be used to perform pharmacological treatment targeting this tissue needs to be considered. Intravitreal injection is a well-established and widely utilized technique for delivering drugs directly to the retina, particularly in the treatment of ocular diseases such as age-related macular degeneration²¹, diabetic retinopathy^22, and retinitis pigmentosa²³...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.3% Triton-X-100	Sigma-Aldrich	T8787	Solution for blocking buffer
1% BSA	Sigma-Aldrich	A4503	Solution for blocking buffer
1% Human BD Fc Block	BD Biosciences	564220	Fc Block required for blocking buffer
150 W LED lamp	Amazon	B07VH3CVSF	LED light source for tissue photoirradiation
1x PBS	Gibco	10010-023	Solution for blocking buffer and wash steps
20 Gallon Storage Container	Amazon	B001B1C4G0	Container for photoirradiation process
40 W RGBW flood LED lamp	Amazon	B08QFPJSMD	RGB light source for tissue photoirradiation
Ag85B	Abcam	ab43019/NA	Antibody for IBEX imaging. See Table 1.
Alpha-smooth muscle actin DyLight 755	Novus Biologicals	NBP2-345221R/NA	Antibody for IBEX imaging. See Table 1.
Avidin/Biotin Blocking Kit	Abcam	ab64212	Avidin and biotin blocking solution
BD Cytofix/Cytoperm	BD Biosciences	554714	Fixation solution
BOND Dewax Solution	Leica Biosystems	NC0221076	Deparaffinization solution
BOND Epitope Retrieval Solution 1	Leica Biosystems	NC0235529	Citrate based pH 6 epitope retrieval solution (ER1)
BOND Epitope Retrieval Solution 2	Leica Biosystems	NC0235530	EDTA based pH 9 epitope retrieval solution (ER2)
BOND Research Detection System	Leica Biosystems	NC0396648	Automated research staining reagent
BOND RX Research Stainer	Leica Biosystems	NA	Automated research staining platform
BOND Wash Solution	Leica Biosystems	NC0221077	Wash buffer
CD15	BD Biosciences	347420/AB_400298	Antibody for IBEX imaging. See Table 1.
CD20 eFluor 660	Thermo Fisher Scientific	50-0202-82/AB_11150959	Antibody for IBEX imaging. See Table 1.
CD4 Alexa Fluor 488	R&D Systems	FAB8165G/AB_2728839	Antibody for IBEX imaging. See Table 1.
CD45 Phycoerythrin (PE)	Novus Biologicals	NBP2-34528PE/NA	Antibody for IBEX imaging. See Table 1.
CD68 iFluor 594	Caprico Biotechnologies	1064135/AB_2892745	Antibody for IBEX imaging. See Table 1.
Donkey anti-goat IgG Alexa Fluor Plus 488	Thermo Fisher Scientific	A32814/AB_2762838	Antibody for IBEX imaging. See Table 1.
Donkey anti-mouse IgG Alexa Fluor Plus AF647	Thermo Fisher Scientific	A32787/ AB_2762830	Antibody for IBEX imaging. See Table 1.
Donkey anti-mouse IgM Alexa Fluor 488	Jackson ImmunoResearch	715-545-020/AB_2340844	Antibody for IBEX imaging. See Table 1.
Donkey anti-rabbit IgG Alexa Fluor Plus AF555	Thermo Fisher Scientific	A32794/AB_2762834	Antibody for IBEX imaging. See Table 1.
EasyDip Staining System	Newcomer Supply	5300KIT	Slide staining system
Fluoromount-G	Thermo Fisher Scientific	00-4958-02	Mounting media
Hoechst	Biotium	40046	Nuclear stain for IBEX imaging. See Table 1.
Imaris File Converter x64 10.2.0	Imaris Oxford Instruments		Free file converter
ImarisViewer software x64 10.2.0	Imaris Oxford Instruments		Free image processing software
ImmEdge Pen	Vector Laboratories	H-4000	Hydrophobic barrier pen
Lithium Borohydride (1 gram)	STREM Chemicals	93-0397	Chemical for dye inactivation, buy small aliquots of this chemical (1 gram)
Lumican Biotin	R&D Systems	BAF2846	Antibody for IBEX imaging. See Table 1.
Micro Cover Glasses	VWR	48393-241	Glass slide covers
Pan-cytokeratin Alexa Fluor 750	Novus Biologicals	NBP2-33200AF750/AB_2868569	Antibody for IBEX imaging. See Table 1.
PELCO BioWave Pro Microwave System	Ted Pella Inc	36500-230	Non-heating scientific microwave
PELCO SteadyTemp Pro	Ted Pella Inc	50062	Fully automated temperature control for scientific microwave
SimpleITK	Open source software		Registration and channel processing software
Streptavidin Alexa Fluor 594	Thermo Fisher Scientific	S11227	Labeling reagent for IBEX imaging. See Table 1.
Widefield Microscope	Leica Microsystems	NA	Leica Microsystems THUNDER imager with LED8 light source and LAS X software (3.7.1.21655). For fluorescence imaging, a custom quad-band filter with external filter wheel (PN: 11536075) with two additional single-band filters (PN: 8118215) were used to image seven dye channels per pass. The filter excitation, dichroic and emission lines are (1) Quad-Band cube: dichroic at 391/32, 479/33, 554/24, 638/31, no excitation or emission filters; (2) external filter wheel position 1: 434/32, position 2: 520/40, position 3: 585/20, position 4: 720/60, position 5: pass-through; (3) single-band 1—585/22 excitation, 594 dichroic, 625/30 emission; (4) single-band 2—635/20 excitation, 647 dichroic, 667/30 emission.
Zeiss Optical Lens Cleaner	Amazon	B00GPVQVCO	Microscope objective lens cleaner
Antibodies
Ag85B	Abcam	ab43019	Polyclonal Rabbit IgG, cycle 1
Alpha-smooth muscle actin DyLight 755	Novus Biologicals	NBP2-345221R	1A4/asm-1, cycle 1
CD4 Alexa Fluor 488	R&D Systems	FAB8165G	Polyclonal Goat IgG, cycle 1
CD8 Alexa Fluor 647	BioLegend	372906	C8/144B, cycle 1
CD68 iFluor 594	Caprico Biotechnologies	1064135	KP1, cycle 1
Donkey anti-goat IgG Alexa Fluor Plus 488	Thermo Fisher Scientific	A32814	Polyclonal Donkey, cycle 1
Donkey anti-mouse IgG Alexa Fluor Plus AF647	Thermo Fisher Scientific	A32787	Polyclonal Donkey, cycle 1
Donkey anti-rabbit IgG Alexa Fluor Plus AF555	Thermo Fisher Scientific	A32794	Polyclonal Donkey, cycle 1
Hoechst	Biotium	40046	Nuclear label, cycle 1
CD15	BD Biosciences	347420	MMA, cycle 2
CD20 eFluor 660	Thermo Fisher Scientific	50-0202-82	L26, cycle 2
CD45 Phycoerythrin (PE)	Novus Biologicals	NBP2-34528PE	2B11 + PD7/26, cycle 2
Donkey anti-mouse IgM Alexa Fluor 488	Jackson ImmunoResearch	715-545-020	Polyclonal Donkey, cycle 2
Lumican Biotin	R&D Systems	BAF2846	Polyclonal Goat IgG, cycle 2
Pan-cytokeratin Alexa Fluor 750	Novus Biologicals	NBP2-33200AF750	AE-1/AE-3, cycle 2
Streptavidin Alexa Fluor 594	Thermo Fisher Scientific	S11227	N/A, cycle 2
Hoechst	Biotium	40046	cycle 2
Solutions
Blocking Buffer (Cycle 1)
Triton-X-100	Sigma-Aldrich	T8787	0.3% by volume
BSA	Sigma-Aldrich	A4503	1% by volume
Human BD Fc Block	BD Biosciences	564220	1% by volume
1x PBS	Gibco	10010-023	97.7% by volume
Primary Antibody Staining Solution #1 (Cycle 1)
Blocking Buffer (Cycle 1)	See above		Top up to volume after adding antibodies
Hoechst	Biotium	40046	1:5000
CD8 AF647	BioLegend	372906	1:10
Primary Antibody Staining Solution #2 (Cycle 1)
Blocking Buffer (Cycle 1)	See above		Top up to volume after adding antibodies
CD4 AF488	R&D Systems	FAB8165G	1:10
Ag85B	Abcam	ab43019/NA	1:100
Secondary Antibody Staining Solution #1 (Cycle 1)
Blocking Buffer (Cycle 1)	See above		Top up to volume after adding antibodies
Donkey Anti-Goat AF Plus 488	Thermo Fisher Scientific	A32814/AB_2762838	1:100
Donkey anti-Rabbit AF Plus 555	Thermo Fisher Scientific	A32794/AB_2762834	1:100
Donkey anti-Mouse AF Plus 647	Thermo Fisher Scientific	A32787/AB_2762830	1:50
Primary Antibody Staining Solution #3 (Cycle 1)
Blocking Buffer (Cycle 1)	See above		Top up to volume after adding antibodies
CD68 iF594	Caprico Biotechnologies	1064135	1:10
a-SMA DL755	Novus Biologicals	NBP2-345221R/NA	1:30
BSA-free Blocking Buffer (Cycle 2)
Triton-X-100	Sigma-Aldrich	T8787	0.3% by volume
Human BD Fc Block	BD Biosciences	564220	1% by volume
1x PBS	Gibco	10010-023	98.7% by volume
Primary Antibody Staining Solution #4 (Cycle 2)
BSA-free Blocking Buffer (Cycle 2)	See above		Top up to volume after adding antibodies
CD15	BD Biosciences	347420	1:30
Lumican Biotin	R&D Systems	BAF2846	1:100
CD45 PE	Novus Biologicals	NBP2-34528PE	1:20
CD20 eFluor 660	Thermo Fisher Scientific	50-0202-82	1:10
Pan-Cytokeratin AF750	Novus Biologicals	NBP2-33200AF750	1:10
Secondary Antibody Staining Solution #2 (Cycle 2)
BSA-free Blocking Buffer (Cycle 2)	See above		Top up to volume after adding antibodies
Donkey anti-Mouse IgM AF488	Jackson ImmunoResearch	715-545-020/AB_2340844	1:100
Strepavidin AF594	Thermo Fisher Scientific	S11227	1:200