Antibody staining for light microscopy

1. High quality:
   

1. 1.Fix tissue in PLP or PEM for 30’.
   

2. 2.Wash 1x10’ in PBS; then 2x10’ in 0.1% saponin/PBS
   Never use Na Azide. Can use 0.01% thimerosol
   

3. 3.1° Ab + 10% goat serum in 0.1% saponin overnight
   

4. 4.Wash 3 x 10’ in saponin
   

5. 5.If using (eg) HRP-conj 2° Ab:
   1.  2° Ab + 10% goat serum in 0.1% saponin 1-2 hrs.
   If using vectastain:
   1.  2° biotin-Ab + 10% serum in saponin 1-2 hr
   2.  Wash 3x10’ in saponin
   3.  “A” (1:100) + “B” (1:100)  + 10% serum in saponin 1-2 hr
   

6. 6.Wash 2x10’ in saponin, then 2x10’ in PBS.
   Wash tissue thoroughly before refixing
   

7. 7.Fix 15’ in 2% glutaraldehyde/PBS; wash 3 x 10’ in PBS
   

8. 8.Develop with 0.5 mg/ml DAB in PBS
   Can add 5µl of 8% NiCl2 solution to 1 ml DAB to enhance contrast
   Stop reaction by transferring discs to PBS with 0.01% Na Azide
   

9. 9.Wash 2x10’ in PBS; mount in Aquamount
   

1. High penetration:
   

1. 1.Fix tissue in 4% paraformaldehyde for 20 min.
   

2. 2.Wash 1x10’ in PBS; then 2x10’ in 0.3% Triton-X100/PBS
   Never use Na Azide. Can use 0.01% thimerosol
   Note that Triton-X100 is the same as NP40
   

3. 3.1° Ab + 10% goat serum in 0.3% Triton-X100 for  2 hrs-overnight; shaking
   

4. 4.Wash 2x 10’ in Triton-X100
   

5. 5.2° Ab + 10% goat serum in 0.3% Triton-X100 2 hrs; shaking
   

6. 6.Wash 2x10’ in 0.3% Triton-X100, then 2x10’ in PBS.
   Wash tissue thoroughly before refixing
   

7. 7.Fix 5’-10’ in 2% glutaraldehyde/PBS
   

8. 8.Develop with 0.5 mg/ml DAB in PBS
   Can add 5µl of 8% NiCl2 solution to 1 ml DAB to enhance contrast
   Stop reaction by transferring discs to PBS with 0.01% Na Azide
   

9. 9.Wash 2x10’ in PBS; mount in Aquamount
   

© 2008 Mount Sinai Medical Center