Immunohistochemistry on sections of organs and pelvic ganglia (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

Janet Keast

Keywords

[‘antibodies’]

Subjects

[‘Immunological techniques’, ‘Imaging’]

Release Date

2017-08-07

Abstract

Immunohistochemistry on sections of organs and pelvic ganglia protocol by the Keast Group

Procedure

See PDF.

In this method, sections are mounted directly on slides for subsequent immunohistochemical staining. All data in GUDMAP has been replicated in at least three embryos/animals and representative images uploaded.

Preparation of tissue for cryosectioning

• Fix tissue in 4% paraformaldehyde (PFA)/0.1M PBS (pH 7.2) overnight at 4°C.

• Wash tissue 3X in 0.1M PBS (pH 7.2) for 10 minutes at room temperature.

• If tissue is to be stored prior to cryosectioning, store in 0.1M PBS (pH 7.2) with 0.1% sodium azide at 4°C.

• Incubate tissues 18-24 h in 30% sucrose/0.1M PBS (pH 7.2) at 4°C.

• Embed tissue in OCT and freeze. Cut sections at 14 µm, mounting onto 1% gelatinised slides.

• Air-dry slides for at least 10 minutes.

Immunohistochemistry

Pre-treatment of sections

• Wash slides 2X in 0.1M PBS (pH 7.2) for 10 minutes at room temperature, shaking gently.

• Dry back of slide with Kimwipe and carefully wipe around sections to remove as much PBS as possible from the sections, but not allowing the sections to dry out.

• Apply 100 µl of blocking solution to each slide and incubate at room temperature in a humid chamber for 1-2 hours.

• Blocking solution: 10% non-immune horse serum (Sigma Cat #12449C) + 0.1% Triton in 0.1M PBS (pH 7.2). This solution should be made up on the day, not stored.

Primary Antibody Application

• Dilute the primary antibody in hypertonic PBS (see below). The optimum dilution of the primary antibody should be determined beforehand in a separate study on tissue of the same species prepared in a similar way.

• Wipe off excess blocking solution from around sections and add ~100 µl of primary antibody to each slide, ensuring sections do not dry out. Incubate 18-24 h at room temperature in a humid chamber.

• Wash slides in 0.1M PBS (pH 7.2) for 15 minutes at room temperature, shaking gently.

Secondary Antibody Application

• Dilute the secondary antibody in hypertonic PBS (see below). The optimum dilution of the secondary antibody should be determined beforehand in a separate study on tissue prepared in a similar way.

• Wipe off excess blocking solution from around sections and add ~100 µl of secondary antibody to each slide, ensuring sections do not dry out. Incubate for 2-3 hours at room temperature in the dark.

• The optimum dilution of the secondary antibody used is determined for each experiment.

Mounting

• Wash slides in 0.1M PBS (pH7.2) for 15 minutes at room temperature, shaking gently.

• Coverslip slides in commercial anti-fade mountant (e.g., Vectashield). Seal edges of coverslip with nail polish.

Solutions

Solutions A and B (below) are used for making two types of PBS:

Solution A: (0.2M): 24.0g NaH2PO4 / 1000 ml H2O

Solution B: (0.2M): 28.4g Na2HPO4 / 1000 ml H2O

Phosphate Buffered Saline (PBS) 0.1M (pH 7.2)

1. 140 ml Sol A + 360 ml Sol B + ~450 ml H2O + 8.5g NaCl. Dissolve.

2. pH to 7.2 with 1M NaOH/HCl

3. Make up to 1 litre with H2O

Hypertonic PBS (pH 7.2)

1. 70 ml Sol A + 180 ml Sol B + ~200 ml H2O + 8.5g NaCl and dissolve

2. pH to 7.2 with 1M NaOH/HCl

3. Make up to 500 ml with H2O

Antibody Lists

Primary antibody list including supplier details

Secondary antibody list including supplier details

Consortium

GenitoUrinary Development Molecular Anatomy Project (GUDMAP) Consortium