TITLE: Protocol for ABC Immunohistochemistry and Quantifying Nerves
AUTHORS: Donald Hoover

[DESCRIPTION]
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. 

[STEPS]
SECTION: Immunostaining Day 1
11. 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.
SECTION: Immunostaining Day 1
12. Rinse slides by placing them into a Coplin jar filled with PBS (pH 7.3 - 7.4): 4 x 5 min each
SECTION: Immunostaining Day 1
13. Place slides in 0.5% BSA + 0.4% Triton X-100 in PBS: 1 x 10 min
SECTION: Immunostaining Day 1
14. Place slides in 1.0% H2O2 in PBS: 1 x 15 min. (2 ml of 30%  H2O2 in 60 ml of PBS)
SECTION: Immunostaining Day 1
15. Place slides in PBS: 4 x 5 min each
SECTION: Immunostaining Day 1
16. Place slides in 0.5% BSA + 0.4% Triton X-100 in PBS: 1 x 10 min
SECTION: Immunostaining Day 1
17. 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.
SECTION: Immunostaining Day 1
18. Place the slides into a black, covered slide incubation box/humidity box
SECTION: Immunostaining Day 1
19. 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)

SECTION: Immunostaining Day 1
20. Allow the sections to remain in blocking buffer for 1.5 - 2 hours at room temperature
SECTION: Immunostaining Day 1
21. Pour off the blocking buffer
SECTION: Immunostaining Day 1
22. Replace with primary antibody solution (antibody of choice diluted in the same blocking buffer used above)


SECTION: Immunostaining Day 1
23. Incubate overnight at room temperature
SECTION: Immunostaining Day 2
24. Place slides in PBS: 4 x 5 min each
SECTION: Immunostaining Day 2
25. Place slides in 0.5% BSA + 0.4% Triton X-100 in PBS: 1 x 10 min
SECTION: Immunostaining Day 2
26. 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.)
SECTION: Immunostaining Day 2
28. Cover the tissue with the secondary antibody solution and incubate for 2 hours at RT in the humidity box
SECTION: Immunostaining Day 2
29. 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.


SECTION: Immunostaining Day 2
30. Pour off the secondary antibody solution
SECTION: Immunostaining Day 2
31. Place slides in PBS: 4 x 5 min each
SECTION: Immunostaining Day 2
32. Place slides in 0.5% BSA + 0.4% Triton X-100 in PBS: 1 x 10 min
SECTION: Immunostaining Day 2
34. Cover the tissue with the ABC reagent and incubate 1-1.5 hrs. in the humidity box
SECTION: Immunostaining Day 2
35. 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)
SECTION: Immunostaining Day 2
36. Pour the ABC reagent off the slides
SECTION: Immunostaining Day 2
37. Place the slides in a Coplin jar with the Tris buffer: 2 x 10 mins.
SECTION: Immunostaining Day 2
38. 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.
SECTION: Immunostaining Day 2
39. Place the slides onto a light-colored background to monitor color development.
SECTION: Immunostaining Day 2
40. Add the VIP color development solution to the tissue sections.
SECTION: Immunostaining Day 2
41. Monitor development of color, which should take from 2 to 20 min to reach maximum intensity. (Use a microscope to monitor.)
SECTION: Immunostaining Day 2
42. Stop color development by submerging slides in dH2O in a Coplin jar
SECTION: Immunostaining Day 2
43. Rinse with dH2O: 2 x 2 min each
SECTION: Immunostaining Day 2
44. Dehydrate the tissue
SECTION: Immunostaining Day 2
44.1. rinse in 50% EtOH: 1x 2 min
SECTION: Immunostaining Day 2
44.2. rinse in 75% EtOH: 1 x 2 min
SECTION: Immunostaining Day 2
44.3. rinse in 95% EtOH: 2 x 2 min
SECTION: Immunostaining Day 2
44.4. rinse in 100% EtOH: 2 x 2 min
SECTION: Immunostaining Day 2
44.5. rinse in Xylene: 2 x 5 min
SECTION: Immunostaining Day 2
45. Attach coverslips using Cytoseal XYL (Thermo Scientific, Cat. No. 8312-4)
SECTION: Immunostaining Day 2
46. Allow to air dry overnight
SECTION: Imaging
47. 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)
SECTION: Immunostaining Day 1
9. 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.


SECTION: Tissue Sectioning using Cryostat
6. Cut tissues into 30μm sections at -20 to -25°C using a Leica CM3050S cryostat (Leica Microsystems Inc., Bannockburn, IL, USA) 
SECTION: Tissue Preparation
2. Tissues shipped to ETSU at 4°C in a PBS solution containing 20% sucrose + 0.02% sodium azide
SECTION: Tissue Preparation
3. Remove tissue samples from solution and dissect as needed, to isolate specific regions
SECTION: Tissue Preparation
4. Freeze tissues on dry ice and store at -80°C until ready to be sectioned
SECTION: Tissue Sectioning using Cryostat
5. Remove tissues from -80°C freezer and mount onto specimen plate using Tissue-Tek® O.C.T. Compound (Sakura Finetek USA, Cat. No. 4583)
SECTION: Tissue Sectioning using Cryostat
7. 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
SECTION: Tissue Sectioning using Cryostat
8. Store sets of tissues in slide boxes wrapped in aluminum foil at -20°C until further processing
SECTION: Imaging
48. 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.
SECTION: Immunostaining Day 1
10. Leave set out to thaw at RT for 20 mins
SECTION: Imaging
50. Save images as JPGs in groups sorted by specimen, region, and stain
SECTION: Image Analysis
55. Record the nerve density percentage shown into a spreadsheet. Repeat the above adjustment until you have three consistent readings
SECTION: Image Analysis
51. Open desired JPG image within the ImageJ program
SECTION: Image Analysis
52. Isolate and "fill" any high-contrast anomalies that should not be included in calculations
SECTION: Image Analysis
53. Change image type to "8-bit."
SECTION: Image Analysis
54. Adjust the threshold of the image until only the desired regions containing nerves are highlighted in red
SECTION: Image Analysis
56. Calculate the average of the three readings
SECTION: Statistical Analysis
57. Input the average nerve density data (% area) into GraphPad Prism 8 for statistical analysis. Data should be separated by donor, heart region, and stain
SECTION: Statistical Analysis
58. Use the "descriptive statistics" function of the software to find the mean and standard deviation of each region
SECTION: Imaging
49. Process each slide using EFI (extended focus image) to enhance Z-stack image.
SECTION: Immunostaining Day 2
27. 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.
SECTION: Immunostaining Day 2
33. 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.
SECTION: Tissue Fixation
1. Tissues fixed in 4% paraformaldehyde in PBS at 4°C for 24 hrs