Jan 12, 2023
Protocol for ABC Immunohistochemistry and Quantifying Nerves
- 1East Tennessee State University
Protocol Citation: Donald Hoover 2023. Protocol for ABC Immunohistochemistry and Quantifying Nerves. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lypoymlx9/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 29, 2021
Last Modified: January 12, 2023
Protocol Integer ID: 54647
Funders Acknowledgement:
NIH SPARC
Grant ID: OT2OD023848
Abstract
Cardiac tissue samples were obtained from human donors and sent to East Tennessee State University for processing. At ETSU, the tissue was dissected, sectioned with a cryostat, and stained for neuronal markers using the ABC IHC method. Pictures of the sections were obtained and analyzed for nerve density across different regions of the heart.
Tissue Fixation
Tissue Fixation
Tissues fixed in 4% paraformaldehyde in PBS at 4°C for 24 hrs
Tissue Preparation
Tissue Preparation
Tissues shipped to ETSU at 4°C in a PBS solution containing 20% sucrose + 0.02% sodium azide
Remove tissue samples from solution and dissect as needed, to isolate specific regions
Freeze tissues on dry ice and store at -80°C until ready to be sectioned
Tissue Sectioning using Cryostat
Tissue Sectioning using Cryostat
Remove tissues from -80°C freezer and mount onto specimen plate using Tissue-Tek® O.C.T. Compound (Sakura Finetek USA, Cat. No. 4583)
Cut tissues into 30μm sections at -20 to -25°C using a Leica CM3050S cryostat (Leica Microsystems Inc., Bannockburn, IL, USA)
Collect sections on charged slides in a sequence that yields at least 4 sets of tissue sections per region of the heart, with each set spanning the entire thickness of the specimen
Store sets of tissues in slide boxes wrapped in aluminum foil at -20°C until further processing
Immunostaining Day 1
Immunostaining Day 1
Stain sets 1, 2, and 3 of each tissue region with a different primary antibody: sheep anti-tyrosine hydroxylase (TH, Milipore Cat. No. AB1542, 1:500) or rabbit anti-tyrosine hydroxylase (TH, Pel-Freez Biologicals, Cat. No. P40101-150, 1:500), rabbit anti-vesicular acetylcholine transporter (VAChT, Synaptic Systems, Cat. No. 139103, 1:500), and rabbit anti-protein gene product 9.5 (PGP9.5, Abcam Cat. No. ab108986, 1:2000), respectively.
Leave set out to thaw at RT for 20 mins
20m
Using the PAP Pen, carefully draw a water barrier circle around the tissue sections on the slide — allow this circle to dry for several seconds or up to approximately one minute.
Rinse slides by placing them into a Coplin jar filled with PBS (pH 7.3 - 7.4): 4 x 5 min each
20m
Place slides in 0.5% BSA + 0.4% Triton X-100 in PBS: 1 x 10 min
10m
Place slides in 1.0% H2O2 in PBS: 1 x 15 min. (2 ml of 30% H2O2 in 60 ml of PBS)
15m
Place slides in PBS: 4 x 5 min each
20m
Place slides in 0.5% BSA + 0.4% Triton X-100 in PBS: 1 x 10 min
10m
Remove slides one at a time and use a clean Kimwipe to wipe around the tissue sections to dry the slide. Keep the sections wet.
Place the slides into a black, covered slide incubation box/humidity box
Cover the tissue sections with blocking buffer (150μl of normal goat serum (Jackson ImmunoResearch Laboratories, Cat. No. 005-000-121) OR normal rabbit serum (Jackson ImmunoResearch Laboratories, Cat. No. 011-000-120) + 10 mL of 1.0% BSA + 0.4% Triton X-100 in PBS)
Allow the sections to remain in blocking buffer for 1.5 - 2 hours at room temperature
2h
Pour off the blocking buffer
Replace with primary antibody solution (antibody of choice diluted in the same blocking buffer used above)
Incubate overnight at room temperature
Immunostaining Day 2
Immunostaining Day 2
6h 6m
6h 6m
Place slides in PBS: 4 x 5 min each
20m
Place slides in 0.5% BSA + 0.4% Triton X-100 in PBS: 1 x 10 min
10m
While rinsing slides above, prepare the biotinylated secondary antibody solution. (Blocking buffer = 150μl of normal goat serum (Jackson ImmunoResearch Laboratories, Cat. No. 005-000-121) OR normal rabbit serum (Jackson ImmunoResearch Laboratories, Cat. No. 011-000-120) + 10 mL of 1.0% BSA + 0.4% Triton X-100 in PBS. Add 50μl of biotinylated secondary antibody to the blocking buffer solution.)
Remove slides one at a time and use a clean Kimwipe to wipe around the tissue sections to dry the slide. Keep the sections wet.
Cover the tissue with the secondary antibody solution and incubate for 2 hours at RT in the humidity box
2h
Prepare the ABC reagent from the Vector Kit at least 30 min prior to using. ABC reagent is made by adding 1 drop of solution A + 1 drop of solution B to 2.5 mL of solution containing 1.0% BSA + 0.4% Triton-X 100 + PBS. Add solutions
A and B to the buffer in that order. Let this sit at RT while performing the following rinses.
Pour off the secondary antibody solution
Place slides in PBS: 4 x 5 min each
20m
Place slides in 0.5% BSA + 0.4% Triton X-100 in PBS: 1 x 10 min
10m
Remove slides one at a time and use a clean Kimwipe to wipe around the tissue sections to dry the slide. Keep the sections wet.
Cover the tissue with the ABC reagent and incubate 1-1.5 hrs. in the humidity box
2h
Prepare 50mM Tris buffer (Trizma, pH 7.6) for the next rinse stage — (1.49 g Trizma (pH 7.6, Sigma T7943) + 200 mL dH2O)
Pour the ABC reagent off the slides
Place the slides in a Coplin jar with the Tris buffer: 2 x 10 mins.
20m
During the last Tris buffer rinse, prepare the VIP development solution — must be at RT for proper use (Vector ImmPACT VIP Kit, SK4605). Use 5 mL VIP diluent + 3 drops of each of solutions 1, 2, 3, and 4 from the kit.
Place the slides onto a light-colored background to monitor color development.
Add the VIP color development solution to the tissue sections.
Monitor development of color, which should take from 2 to 20 min to reach maximum intensity. (Use a microscope to monitor.)
20m
Stop color development by submerging slides in dH2O in a Coplin jar
Rinse with dH2O: 2 x 2 min each
4m
Dehydrate the tissue
rinse in 50% EtOH: 1x 2 min
2m
rinse in 75% EtOH: 1 x 2 min
2m
rinse in 95% EtOH: 2 x 2 min
4m
rinse in 100% EtOH: 2 x 2 min
4m
rinse in Xylene: 2 x 5 min
10m
Attach coverslips using Cytoseal XYL (Thermo Scientific, Cat. No. 8312-4)
Allow to air dry overnight
Imaging
Imaging
6h 6m
6h 6m
Image tissue sections using Olympus BX41 microscope equipped with an Olympus DP74 digital camera and cellSens software (Olympus America Inc., Center Valley, PA; RRID:SCR_016238)
Load slide onto stage and collect representative Z-stack images of each slide at 20X magnification. The Z-stack range is defined by the researcher as the furthest depth at which some nerves are still in focus, and the step size is determined by the program.
Process each slide using EFI (extended focus image) to enhance Z-stack image.
Save images as JPGs in groups sorted by specimen, region, and stain
Image Analysis
Image Analysis
6h 6m
6h 6m
Open desired JPG image within the ImageJ program
Isolate and "fill" any high-contrast anomalies that should not be included in calculations
Change image type to "8-bit."
Adjust the threshold of the image until only the desired regions containing nerves are highlighted in red
Record the nerve density percentage shown into a spreadsheet. Repeat the above adjustment until you have three consistent readings
Calculate the average of the three readings
Statistical Analysis
Statistical Analysis
Input the average nerve density data (% area) into GraphPad Prism 8 for statistical analysis. Data should be separated by donor, heart region, and stain
Use the "descriptive statistics" function of the software to find the mean and standard deviation of each region