Antibody Staining Protocol on Frozen Kidney 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

Thomas Carroll; Alicia Fessler

Keywords

[‘antibodies’, ‘immunofluorescence’, ‘immunofluorescent’, ‘kidney’, ‘mouse’, ‘section’]

Subjects

[‘Molecular biology’]

Release Date

2020-08-20

Abstract

Antibody Staining Protocol on 10um Frozen Kidney Sections

Reagents

• Triton x-100 (Sigma, 1086431000)
• 1M Tris pH 7.5 (Sigma, 11814273001)
• 0.5M EDTA pH 8.0 (Sigma, ED)
• Sodium Citrate (Sigma, S4641)
• Citric Acid (Sigma, 251275)
• Pap pen (Sigma, Z377821)
• heat inactivated FBS
• 1x PBS pH 7.4
• DAPI (ThermoFisher Scientific, D1306)
• Vecta Sheild (Vector Laboratories, H-1000)
• Primary antibody (assay specific)
• Secondary antibody (assay specific)

Equipment

• Nutator placed at room temperature
• 2L Beaker
• Microwave
• Slide box
• Coverslip

Procedure

1. Wash in PBS pH 7.4 with 0.1% Trition for 5 minutes x 3 times on nutator placed at room temperature

2. Antigen retrieval: make either TE or citrate in a 2L beaker (antibody dependent). Cover and microwave for 17 minutes. Cool on ice until slides are room temperature.

TE antigen retrieval

• 1mL 1M Tris pH 7.5
• 10mL 0.5M EDTA pH 8.0
• adjust total volume to 1L with miliQ water

Citrate antigen retrieval

• 2.94g sodium citrate
• 1L miliQ water
• pH to 6.0 using 1M citric acid

1. Wash in PBS pH 7.4 with 0.1% Trition for 5 minutes x 1 time on nutator place at room temperature

2. Pap pen slides

3. Block for 1 hour at room temperature in 5% heat inactivated FBS in 0.1% Triton in PBS pH 7.4 in a humidified slide box

4. Add primary antibody. Keep in 4C overnight.

5. Remove and save primary antibody. Wash in PBS pH 7.4 with 0.1% Trition for 5 minutes x 3 times on nutator placed at room temperature

6. Re-pap pen slides

7. Add secondary antibody diluted 1:500 in block for 1 hour at room temperature.

8. Wash in PBS pH 7.4 with 0.1% Trition for 5 minutes x 3 times on nutator placed at room temperature

9. Add DAPI diluted 1:1000 in MQ for 10 minutes at room temperature

10. Wash in PBS pH 7.4 with 0.1% Trition for 5 minutes x 3 times on nutator placed at room temperature

11. Mount slides with one drop of Vecta shield without DAPI avoiding bubbles. Store in a slide folder in -20C.

Consortium

(Re)Building a Kidney (RBK) Consortium