Immunohistochemistry Protocol (Mouse Adult Tissue)
(for Calbindin D-28K ICC)
Modified for Hongbin’s ICC exp. (Jul. 26, 2005)
Materials:
PBS: phosphate-buffered saline, pH 7.6
BSA: bovine serum albumin, 0.1%
NGS: normal goat serum, 3%
0.05% H2O2
0.5% Triton X-100
0.05M Tris buffer, pH 7.6
1ºAb: Mouse anti-Calbindin D-28K and
2ºAb: Goat anti-Mouse (biotinylated) IgG
ABC solution (Vector Elite Kit)
Methanol
Petri Dishes (for mounting)
Small paint brush
Small ICC dishes (12 wells/dish)
Day 1.
1. Experiment done, in most cases, in room temperature.
2. If sections are in solution other than PBS rinse sections in PBS/TX-100.
3. Incubate for 20 min in the following solution:
For 3 ml/well: 10% Methanol 300 ul
0.1% BSA 3 mg
3% NGS 90 ul
0.05% H2O2 (3%) 15 ul
0.5%Triton x-100/PBS 2.6 ml
4. Rinse in PBS/TX-100 until no further bubbles.
5. Incubate in 1ºAb (Mouse anti-Calbindin D-28K) in PBS/TX-100 containing 1% NGS overnight in refrigerator for 48 hrs on Rocker.
1ºAb: (for 3 ml) Mouse anti-Calbindin D-28K (1:2000) 1.5 ul
NGS (1%) 30 ul
TX-100/PBS 3 ml
Day 2.
6. Rinse samples in TX-100/PBS for 5-10 min x3 (1ºAb can be stored frozen).
7. Incubate in 2ºAb (biotinylated Goat anti-mouse IgG 1:1000) in PBS containing 1% NRS for 2 hour at room temp.
2ºAb: (for 3 ml) Goat anti-mouse IgG (1:1000) 3 ul
NGS (1%) 30 ul
TX-100/PBS 2.9 ml
8. Rinse with PBS/TX-100 for 5-10 min x3.
9. Incubate with Biotin/Avindin kit 15 ul each of solutions A & B in 3 ml PBS/TX/1% NGS (prepared 30 min prior to use) for 30 min.
10. Rinse with Tris buffer for 5-10 min x3.
11. Incubate in DAB/Tris for 10 min (4-5 mg/10 ml Tris). (for 3 ml soln, need 100x DAB 30 ul)
12. Add 9 ul of 3% H2O2/3 ml, reapply to sample for 30 sec or more.
13. Stop reaction with Tris. (DO STEPS 11 & 12 ONE TIME FOR ONE WELL ONLY)
14. Rinse sections well with Tris.
15. Rinse sections 5 times x 5 min with PBS
16. Mount the sections onto gelatin coated glass slides in order, and air dry sections for 2-3 days.
Day 3.
17. Dehydrate the sections in increasing order of ethanol (10 min each) and then in Histoclear for two times (15 min each).
18. Coverslip sections and put them on board for air-drying (several days).