Fluorescent Immunocytochemistry Protocol on Frozen sections (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Kevin Gaido; Janan Hensley

Keywords

[‘immunofluorescence’]

Subjects

[‘Cell biology’, ‘Immunological techniques’]

Release Date

2017-08-07

Abstract

Fluorescent Immunocytochemistry Protocol on Frozen sections by the Gaido Group

Procedure

1. Prepare cryostat section at 5-7 mm (for In situ, may 10 mm)

2. Fix with 4% paraformaldehyde (diluted 16% paraformaldehyde at 1:4 in PBS), 10 min, room temperature

3. Wash with PBS/Triton, 5 min X 3

4. Block with Blocking buffer (500 ml normal goat serum to 10 ml PBS + 10 ml Triton, serum match to species the second antibody from), 1 hr, room temperature

5. Wash with PBS, 5 min X 1

6. Primary antibody diluted in 3% BSA (300mg in 10 ml PBS), overnight, at 4 °C

7. Wash with PBS, 5 min X 3

8. Incubate with biotinylated secondary antibody, 1:500 dilution (pay attention to different species, against primary), 1 hr, room temperature

9. Wash with PBS, 5 min X 3

10. Mount coverslips with VectaMount

11. Visualization using microscope

12. Analysis using Confocal Software

Consortium

GenitoUrinary Development Molecular Anatomy Project (GUDMAP) Consortium