Immunofluorescence

Immunostaining on frozen sections Beatriz Sosa-Pineda Lab

Tissue Preparation

1. Dissect tissues, rinse with cold PBS and fix overnight with 4% PFA (al least  5x volumes) in PBS, at 4°C.
2. Transfer tissues to 30% sucrose in PBS (at least 5x volumes) and rock overnight at 4°C.
3. Cover tissues with Tissue-Tek (OCT) and freeze on dry ice.
4. Section the specimens with a cryostat (10 µm). Sections can be stored at – 20°C or processed immediately as follows:

1st Day

1. Allow slides to warm to RT for 5-10 minutes.

2. Outline sections with hydrophobic pen.

3. Wash with TBS-T (TBS + 0.05%Tween 20) for 10 min on rocker.

4. Place slides in humidified chamber and block for 30 min @ RT with blocking solution.

(Blocking solution:

1. Maleate buffer: 100 mM maleic acid, 150 mM NaCl.
2. Blocking reagent (Roche cat No 11 096 176 001.) Dissolve the blocking reagent in maleate buffer to a final concentration of 10%, store it @ -20°C as 10 ml aliquots.
3. Fetal bovine serum

Prepare blocking solution by mixing 3 mL of Maleate buffer, 1 mL of FBS and 1 mL of 10% blocking reagent.)

5. Remove blocking solution and add primary Antibody diluted in blocking solution to the sections, and incubate O/N in the humidified chamber at RT.

RbGS (abcam cat no ab49873): 1/5000;

RatEcad (Thermo Fisher Scientific / Life Technologies cat no 13-1900): 1/5000 

2nd Day

6. Remove primary antibody and wash 3X 10 min with TBS-T on rocker.

7. Incubate with fluorescent labeled Secondary Antibody, diluted in blocking solution, for 2-3 h @ RT.

1/250 dilution

8. Remove antibody and wash 3X 5 min with PBS on rocker.

9. Incubate the slides with DAPI (1:1,000 dilution) for 5 min @ RT

10. Wash 1X 5 min PBS

11. Wash 2X quickly in dH2O (“Dip and remove”)

12. Mount with Prolong Gold and coverslip

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