Protocol for ABC Immunohistochemistry and Quantifying Nerves

Tissues fixed in 4% paraformaldehyde in PBS at 4°C for 24 hrs

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

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

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

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

Place slides in 0.5% BSA + 0.4% Triton X-100 in PBS: 1 x 10 min

Place slides in 1.0% H₂O₂ in PBS: 1 x 15 min. (2 ml of 30% H₂O₂ in 60 ml of PBS)

Place slides in PBS: 4 x 5 min each

Place slides in 0.5% BSA + 0.4% Triton X-100 in PBS: 1 x 10 min

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

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

Place slides in PBS: 4 x 5 min each

Place slides in 0.5% BSA + 0.4% Triton X-100 in PBS: 1 x 10 min

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

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

Place slides in 0.5% BSA + 0.4% Triton X-100 in PBS: 1 x 10 min

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

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.

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.)

Stop color development by submerging slides in dH₂O in a Coplin jar

Rinse with dH₂O: 2 x 2 min each

Dehydrate the tissue

rinse in 50% EtOH: 1x 2 min

rinse in 75% EtOH: 1 x 2 min

rinse in 95% EtOH: 2 x 2 min

rinse in 100% EtOH: 2 x 2 min

rinse in Xylene: 2 x 5 min

Attach coverslips using Cytoseal XYL (Thermo Scientific, Cat. No. 8312-4)

Allow to air dry overnight

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

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

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