TITLE: Immunohistochemistry Protocol for Free-floating Fixed Tissue
AUTHORS: Jeffrey Kordower, Yaping Chu

[DESCRIPTION]
Immunohistochemistry protocol for staining free-floating fixed tissue in the Kordower Laboratory.

[GUIDELINES]
HISTO- NOTES:
Primate tissue staining dishes use 100 mLsolution per dish
Rodent tissue staining dishes 50 mLsolution per dish
If staining a large number of primate cases, incubate 1' & 2' Ab in individual cups to conserve volume of Ab used. 
Prepare bleach neutralizing solution prior to Step 12.
Be conscious of tissue saturation while washing and incubating. i.e. Check that tissue is fully submerged in solution & not clumping. This will ensure proper penetration of antibodies & other reagents. 
Always include Positive & Negative Controls.
Positive: Use relevant control tissue to confirm specific antibody detection. (i.e. pS129; control tissue should consist of nigral sections previously successfully stained for pS129).
Negative: Ideally, use tissue that you know does not contain the targeted antigen. If not available, use a section of tissue not incubated in the 1' Ab (primary delete).
When incubating 1' Ab overnight, leave on shaker in refrigerator. 
Can incubate in fridge on a shaker, covered in parafilm, over the weekend or up to 3 days.
Select a secondary antibody directed against the species in which the primary antibody was raised (i.e. if a primary antibody raised in rabbit is used, an anti-rabbit secondary antibody raised in a species other than rabbit must be used).

[STEPS]
SECTION: DAY 1 (4 hrs)
1. Wash sections (6 x 10 min) in Dilution Media (DM) (0.2 Molarity (M)TBS plus 0.05 % volumeTriton X-100).
SECTION: DAY 1 (4 hrs)
2. Endogenous peroxidase inhibition (20 min). 0.1 Molarity (M) Sodium meta-periodate in TBS.
100 mL 0.2 Molarity (m)Tris-buffered saline (TBS)
2.13 g Sodium meta-periodate
SECTION: DAY 1 (4 hrs)
3. Wash (2 x 10 min) in DM.
SECTION: DAY 1 (4 hrs)
4. Serum blocking step (60 min incubation):
100 mL DM
3 mL Normal Serum (species matching the host of the secondary antibody, e.g. horse, goat)
2 g Bovine Serum Albumin (BSA) 
SECTION: DAY 1 (4 hrs)
5. Incubation in primary antibody (1080 min- 4320 min). See antibody catalog for concentration of primary antibody.
100 mL DM 
1 mL Normal Serum (species matching the host of the secondary antibody, e.g. horse, goat)
1 g BSA
0.5 mL Triton X-100
 

SECTION: DAY 2 (8 hrs)
7. Secondary antibody incubation (60 min) Concentration of secondary antibody is always 1:200 in solvent.
100 mL DM
1 mL Normal Serum (species matching the host of the secondary antibody, e.g. horse, goat)
1 g BSA
SECTION: DAY 2 (8 hrs)
8. Wash (6 x 10 min) in DM.
 

SECTION: DAY 2 (8 hrs)
9. Avidin-Biotin Complex (ABC) Step (60 min) - Vectastain Elite ABC-HRP Kit (PK-6100).
100 mL DM
1 mL Normal Serum (species matching the host of the secondary antibody, e.g. horse, goat) 
1 g BSA
SECTION: DAY 2 (8 hrs)
9.1. Add ABC Reagent A and B to 1/10th of total desired volume of solvent.
SECTION: DAY 2 (8 hrs)
10. Wash for 10 min in DM.
SECTION: DAY 2 (8 hrs)
9.2. Incubate for 30 min at Room temperature. Then dilute 1:10 using the same solvent.
 
 
     Working Solution      A (drops)      B (drops)      1/10th Working solution        25 mL      1      1      2.5 mL        50 mL      2      2      5 mL        100 mL      4      4      10 mL   

SECTION: DAY 2 (8 hrs)
11. Wash for 10 min with TBS.
SECTION: DAY 2 (8 hrs)
12. Wash (3 x 10 min) in 0.2 Molarity (M) Imidazole/1.0 Molarity (M) Sodium Acetate buffer pH 7.2 to pH 7.4 .
1000 mL dH2O 
0.68 g Imidazole 
6.8 g Sodium Acetate. 
Retain 100 mL of non-pH’d buffer for DAB preparation. 
SECTION: DAY 2 (8 hrs)
13. DAB Step (Neutralize DAB with bleach when done)
SECTION: DAY 2 (8 hrs)
14. Wash developed tissue in imidazole acetate buffer (3 x 10 min).

 

SECTION: DAY 2 (8 hrs)
6. Wash (6 x 10 min) in DM.
SECTION: DAY 2 (8 hrs)
6.1. Wash in DM for 10 min (1/6).
SECTION: DAY 2 (8 hrs)
6.6. Wash in DM for 10 min (6/6).
SECTION: DAY 2 (8 hrs)
6.5. Wash in DM for 10 min (5/6).
SECTION: DAY 2 (8 hrs)
6.4. Wash in DM for 10 min (4/6).
SECTION: DAY 2 (8 hrs)
6.3. Wash in DM for 10 min (3/6).
SECTION: DAY 2 (8 hrs)
6.2. Wash in DM for 10 min (2/6).
SECTION: DAY 2 (8 hrs)
8.1. Wash for 10 min in DM (1/6).
SECTION: DAY 2 (8 hrs)
8.5. Wash for 10 min in DM (5/6).
SECTION: DAY 2 (8 hrs)
8.4. Wash for 10 min in DM (4/6).
SECTION: DAY 2 (8 hrs)
8.3. Wash for 10 min in DM (3/6).
SECTION: DAY 2 (8 hrs)
8.2. Wash for 10 min in DM (2/6).
SECTION: DAY 2 (8 hrs)
8.6. Wash for 10 min in DM (6/6).
SECTION: DAY 1 (4 hrs)
3.1. Wash for 10 min in DM (1/2).
SECTION: DAY 1 (4 hrs)
3.2. Wash for 10 min in DM (2/2).
SECTION: DAY 2 (8 hrs)
12.1. Wash for 10 min in Imidazole/Sodium Acetate buffer (1/3).
SECTION: DAY 2 (8 hrs)
12.3. Wash for 10 min in Imidazole/Sodium Acetate buffer (3/3).
SECTION: DAY 2 (8 hrs)
12.2. Wash for 10 min in Imidazole/Sodium Acetate buffer (2/3).

SECTION: DAY 1 (4 hrs)
1.1. Wash sections for 10 min in DM (1/6).
SECTION: DAY 1 (4 hrs)
1.6. Wash sections for 10 min in DM (6/6).
SECTION: DAY 1 (4 hrs)
1.5. Wash sections for 10 min in DM (5/6).
SECTION: DAY 1 (4 hrs)
1.4. Wash sections for 10 min in DM (4/6).
SECTION: DAY 1 (4 hrs)
1.3. Wash sections for 10 min in DM (3/6).
SECTION: DAY 1 (4 hrs)
1.2. Wash sections for 10 min in DM (2/6).
SECTION: DAY 2 (8 hrs)
13.1. Make DAB solution 

100 mL non-pH'd imidazole acetate buffer from above 
50 mg 3,3-Diaminobenzidine Tetrahydrochloride (DAB)
2 g Nickel(II) sulfate hexahydrate **(Only used with certain primary antibodies, chromagen enhancer that changes brown DAB precipitate to blue-purple)**
SECTION: DAY 2 (8 hrs)
13.5. To monitor signal, move all tissue to imidazole buffer, remove one section and mount on an UNSUBBED slide and view under microscope. Place all tissue back in DAB solution to increase signal intensity, if needed.
SECTION: DAY 2 (8 hrs)
13.4. Place tissue in DAB solution.
Develop tissue for approximately 4 min to 7 min.
Timing is critical, ensure all tissue spends the same amount of time in DAB solution.
SECTION: DAY 2 (8 hrs)
13.3. Start reaction -- add 500 µL of 1 % (v/v) hydrogen peroxide (H2O2) to the above DAB mixture just prior to use.
OR add 16.7 µL of 30 % (v/v) hydrogen peroxide (H2O2), per 100 mL.
SECTION: DAY 2 (8 hrs)
13.2. Make 1 % (v/v) Hydrogen Peroxide (H2O2)

3 mL of dH2O
100 µL of 30 % (v/v) hydrogen peroxide (H2O2)
SECTION: DAY 2 (8 hrs)
14.1. Wash developed tissue in imidazole acetate buffer for 10 min (1/3).
SECTION: DAY 2 (8 hrs)
14.2. Wash developed tissue in imidazole acetate buffer for 10 min (2/3).
SECTION: DAY 2 (8 hrs)
14.3. Wash developed tissue in imidazole acetate buffer for 10 min (3/3).
SECTION: DAY 2 (8 hrs)
15. Store tissue in 0.2 Molarity (m) Phosphate-buffered saline (PBS) in refrigerator 4 °C until mounted on slides.