TITLE: Immunofluorescence Multi-label Protocol for Free-floating Fixed Tissue
AUTHORS: Jeffrey Kordower, Yaping Chu

[DESCRIPTION]
Immunofluorescence multi-label 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. 
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. Fluorophore-conjugated 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 (2 x 10 min) in TBS.


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 TBS (1/2).
SECTION: DAY 2 (8 hrs)
8.2. Wash for 10 min in TBS (2/2).
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)
9. Serum blocking step (60 min incubation):
100 mL DM
5 % (v/v) Normal Serum (species MATCHING THE HOST OF THE PRIMARY antibody, saturates open binding sites on secondary antibody)
1 g Bovine Serum Albumin (BSA)
 

SECTION: DAY 2 (8 hrs)
10. Oversaturate with Fab antibody against host of primary antibody and from same host
species as secondary antibody. (ex. If primary was mouse and secondary was
goat anti-mouse, you would use a Fab-goat anti-mouse antibody). Working
concentration: 40 µg/mL. (60 min incubation)
 100 mL TBS
40 µg/mL Fab antibody (base concentration is 1.3 mg/mL for fab-goat anti mouse, so use M1V1 = M2V2 to find V1/X).
SECTION: DAY 2 (8 hrs)
11. Wash (2 x 10 min) in TBS.


SECTION: DAY 2 (8 hrs)
11.1. Wash for 10 min in TBS (1/2).
SECTION: DAY 2 (8 hrs)
11.2. Wash for 10 min in TBS (2/2).
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 3 (2 hrs)
13. Wash (6 x 10 min) in TBS.
SECTION: DAY 3 (2 hrs)
13.1. Wash in TBS for 10 min (1/6).
SECTION: DAY 3 (2 hrs)
13.2. Wash in TBS for 10 min (2/6).
SECTION: DAY 3 (2 hrs)
13.3. Wash in TBS for 10 min (3/6).
SECTION: DAY 3 (2 hrs)
13.4. Wash in TBS for 10 min (4/6).
SECTION: DAY 3 (2 hrs)
13.5. Wash in TBS for 10 min (5/6).
SECTION: DAY 3 (2 hrs)
13.6. Wash in TBS for 10 min (6/6).
SECTION: DAY 3 (2 hrs)
14. Fluorophore-conjugated secondary antibody incubation against second (and third, if used) primary antibody. (60 min) Use different fluorophores for each secondary. 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 3 (2 hrs)
15. Wash (3 x 10 min) in TBS.


SECTION: DAY 3 (2 hrs)
15.1. Wash for 10 min in TBS (1/3).
SECTION: DAY 3 (2 hrs)
15.2. Wash for 10 min in TBS (2/3).
 

SECTION: DAY 3 (2 hrs)
15.3. Wash for 10 min in TBS (3/3).
SECTION: DAY 2 (8 hrs)
12. Incubation in SECOND 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
 

SECTION: DAY 3 (2 hrs)
16. Store tissue in TBS in the refrigerator at 4 °C until mounted.
SECTION: DAY 3 (2 hrs)
17. Control for Fragment antibody (Fab): Control tissue should be processed alongside experimental tissue through Day 2, Step 11. Skip second primary incubation all together (Step 12), and complete Day 3. Check under microscope to ensure there is no co-labeling between the two chosen fluorophores.
Use appropriate +/- controls.