TITLE: IHC analysis of brain regions from PCB exposed rats
AUTHORS: Amanda Bullert, Morgan Linahon, Michael Dailey, Hansjoachim Lehmler

[DESCRIPTION]
This protocol describes a method to analyze the effect of PCB52 inhalation on brain cells in adolescent rats. PCB52 (2,2’,5,5’-tetrachlorobiphenyl) was administered to adolescent rats via a nose-only apparatus with PCB52-laden air. The exposure was 4 hours a day for 28 consecutive days. Brains were collected, the cerebellum region was frozen, and tissue sections were cut and stained to label apoptotic cells using PSvue, microglia using anti-IBA1, and astrocytes using anti-GFAP. Using a Leica TCS SP8 confocal microscope, Z-Stack images were taken and analyzed for differences in cell density and apoptosis within the distinct cerebellar layers. 

[BEFORE_START]
Note that this protocol involves work with experimental animals and requires prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.

[STEPS]
SECTION: Extraction and preparation of cerebellum brain region
1. Using a rat brain matrix, the brain is cut into roughly 12 sections, each 2 mm thick
SECTION: Extraction and preparation of cerebellum brain region
2. Drop fix the individual sections in 2 mL of 4% formalin per well for 24 hours at 4°C
SECTION: Extraction and preparation of cerebellum brain region
3. Remove the formaldehyde and add 2 mL of 30% sucrose to each well for 24 hours at 4°C
SECTION: Extraction and preparation of cerebellum brain region
4. Place a drop of O.C.T. compound in a disposable plastic cryomold (or any flat surface specimen block) and place the tissue flat on top of the O.C.T. compound
SECTION: Extraction and preparation of cerebellum brain region
6. Place the mold on dry ice to freeze the O.C.T. compound
SECTION: Extraction and preparation of cerebellum brain region
7. Store molds in a -80°C degree freezer 

SECTION: Cutting and staining
8. Set the temperature of the Leica CM3050 S Cryostat to -18°C and set to a thickness to 100 µm
SECTION: Cutting and staining
9. Place O.C.T. tissue molds in the cryostat and wait at least 30 minutes for the blocks to acclimate to the new temperature 
SECTION: Cutting and staining
10. Preload 12-well plate with 1-2 mL of PBS solution (optional: prevent contamination by adding sodium azide)
SECTION: Cutting and staining
11. Cut tissues using the cryostat and transfer slices to the 12-well plate, one slice per well (dispose of excess O.C.T. block as hazardous waste)
SECTION: Cutting and staining
12. Wash with 2 mL of PBS solution 3 times for 15 min at room temp
(Washing includes switching the current solution for fresh PBS solution 3 times, waiting 15 mins in between, and being careful not to disturb the tissues in the wells. During each of the 15-minute intervals, place the well plate on a rotator at around 100 rpm)
SECTION: Cutting and staining
13. In a new 12-well plate, add 0.5 mL of extraction solution to each labeled well
SECTION: Cutting and staining
15. Leave the tissue slices in the extraction solution for 1 hour on the rotator at around 100 rpm at room temp
SECTION: Cutting and staining
16. Replace the extraction solution with 0.5 mL fresh extraction solution in each well
SECTION: Cutting and staining
18. Remove extraction solution by washing slices with 0.5 mL ammonium chloride solution for 30 min at room temp
SECTION: Cutting and staining
19. Replace ammonium chloride solution with 0.5 mL of blocking solution for 30 min at room temp
SECTION: Cutting and staining
20. Replace blocking solution with 0.5 mL antibody incubation solution (1% serum) for 5 min 
SECTION: Cutting and staining
21. Switch the 1% serum solution for ~300 μL of the primary antibody solution (enough to submerge the tissue; make fresh with each batch)
Primary antibody solution (1 mL total)
- 1 μL anti-IBA1 (1:1000)
- 1 μL anti-GFAP (1:1000)
- 9993 μL antibody incubation solution (1% serum)
SECTION: Cutting and staining
24. Wash with antibody incubation solution (1% serum) 3 times for 15 min at room temp
(Washing includes switching the current solution for fresh antibody incubation solution 3 times, waiting 15 mins in between, and being careful not to disturb the tissues in the wells. During each of the 15-minute intervals, place the well plate on a rotator at around 100 rpm)
SECTION: Cutting and staining
25. Switch the 1% serum solution for ~300 μL secondary antibody solution (enough to submerge tissue; make fresh with each batch)
Secondary antibody solution (1 mL total)
- 2 μL Alexa 488 (Goat anti Rabbit) (1:500)
- 2 μL Alexa 647 (Mouse anti Rat) (1:500)
- 9996 μL antibody incubation solution (1% serum)
SECTION: Cutting and staining
28. Wash with 1% serum 3 times for 15 min at room temp
(Washing includes switching the current solution for fresh 1% serum solution 3 times, waiting 15 mins in between, and being careful not to disturb the tissues in the wells. During each of the 15-minute intervals, place the well plate on a rotator at around 100 rpm)
SECTION: Cutting and staining
29. Switch the 1% serum solution for 300 μL of DAPI solution (make fresh with each batch)
DAPI solution (5 mL total)
- 1 μL DAPI (1:5000)
- 4999 μL antibody incubation solution (1% serum)
SECTION: Cutting and staining
31. Wash with 1% serum 3 times for 15 min at room temp
(Washing includes switching the current solution for fresh 1% serum solution 3 times, waiting 15 mins in between, and being careful not to disturb the tissues in the wells. During each of the 15-minute intervals, place the well plate on a rotator at around 100 rpm)
SECTION: Cutting and staining
32. Switch the 1% serum solution for 300 μL of PSVue solution (make fresh with each batch)
PSVue solution (1 mL total)
- 1 μL PSVue550(1:1000)
- 9999 μL antibody incubation solution (1% serum)
SECTION: Cutting and staining
34. Mount on microscope slide with 1-2 drops of PBS with 0.02% sodium azide, flattening the tissue as much as possible before applying the coverslip
SECTION: Imaging and analyzing
37. Place the microscope slide face down on the inverted Leica TCS SP8 Confocal Microscope
SECTION: Imaging and analyzing
39. Adjust the settings, which include: 20x scanning objective, line averaging set to 16 (or as needed), and 1024 x 1024 scan format
SECTION: Imaging and analyzing
38. Using the bright field setting, manually focus the microscope 
SECTION: Imaging and analyzing
40. Once focused, take a full overview image to find the area of interest 
SECTION: Imaging and analyzing
41. Create a sequential setting that includes channels corresponding to the stains used
SECTION: Imaging and analyzing
42. Pan through each channel and adjust the lasers and detectors so the cells are not fluorescently oversaturated but just bright enough for analysis
SECTION: Imaging and analyzing
43. Once each channel is adjusted, take a Z stack 70 μm thick, with a Z step size of 2 μm:

 
SECTION: Imaging and analyzing
44. Once the Z stacks are saved, open them in ImageJ
SECTION: Imaging and analyzing
45. Looking at each channel individually, analyze cell density and % area counts for each stain. The following criteria are to be followed for each stain:

-IBA1: particle size of 15 μm2-infinity, threshold set from 50-60 to 255
-GFAP: particle size of 10 μm2-infinity, threshold set from 50 to 255
-DAPI: particle size of 5 μm2-infinity, threshold set from 50 to 255
-PSVue: particle size of 2 μm2-infinity, threshold set from 100 to 255

(If desired, these parameters can be analyzed for both the molecular and granular layers within the cerebellum)
SECTION: Imaging and analyzing
46. Record the measurements and transfer data to an Excel spreadsheet for analysis and data visualization
SECTION: Imaging and analyzing
47. Channels can be combined to create an overlapping image showing all cell types. Brightness and contrast may need to be adjusted to properly see all of the stains. Representative images are shown below:

 

 

SECTION: Cutting and staining
14. Transfer slices to the new 12-well plate containing extraction solution using a very fine paintbrush to pick up the tissue slices
SECTION: Cutting and staining
17. Incubate tissue slices overnight (approximately 16 hours) in a fridge at 4°C
SECTION: Cutting and staining
23. Incubate the 12-well plates with the tissue slices for 3 nights in a fridge at 4°C
SECTION: Cutting and staining
22. Wrap the plate with parafilm
SECTION: Cutting and staining
27. Incubate tissue slices overnight (approximately 16 hours) in a fridge at 4°C
SECTION: Cutting and staining
26. Wrap the 12-well plate with parafilm, followed by aluminum foil to avoid exposure of the fluorescent probe to light
SECTION: Cutting and staining
30. Incubate tissue slices on a rotator for 30 min at room temp
SECTION: Cutting and staining
33. Incubate tissue slices for at least 2 hours in a fridge at 4°C
SECTION: Cutting and staining
35. Secure the coverslip with 2 layers of clear nail polish
SECTION: Extraction and preparation of cerebellum brain region
5. Fill the rest of the cryomold with O.C.T. compound so it completely covers the tissue
SECTION: Cutting and staining
36.