Immunostaining FFPE Human Kidney Tissue (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

Nikhil Singh

Keywords

[‘human renal cortex’, ‘human’, ‘antibodies’, ‘immunofluorescence’, ‘immunofluorescent’, ‘immunohistochemistry’]

Subjects

[‘Cell biology’, ‘Molecular biology’]

Release Date

2018-02-07

Abstract

This is a protocol optimized for immunostaining of formalin fixed, paraffin embedded human kidney tissue. We have used it with great success for immunofluorescence as well as imaging mass cytometry.

Introduction

Fresh cortex from surgical nephrectomies is fixed in 10% formalin for 24 hours followed by sequential dehydration into 70% Ethanol. Tissue is paraffin-embedded and then sectioned to 5 um sections before being affixed to glass slides.

Reagents

TBS 50mL 1M Tris (final: 50 mM) 8.76g NaCl (final: 150 mM NaCl) Bring final volume to 1L, pH 7.6

TBS 0.05% Tween-20 950mL TBS 50mL Tween-20

TBS/0.05% Tween / 0.3% BSA 500cc TBS 0.05% Tween 1.5g BSA Filter through 0.45um micron filter

1.25mM EDTA 0.74g EDTA (or 5.06 mL of 0.5M EDTA) In 2L ddH2O pH titrated to 8.0

Equipment

Hot plate Kim wipes Hydrophobic marking pen FC blocker #564220 BD ph FBS heat inactivated (incubated at 56C x 30min prior to dlution in blocking solution)

Procedure

Deparaffinize 5 micron sections (60 degrees C overnight) Incubate in old xylenes for 20 mins Incubate in fresh xylenes for 20 mins Rehydration in 100% EtOH twice, then 70% EtOH, then 50% EtOH, then 25% EtOH, then water twice Antigen retrieval in 1.25 mM EDTA pH 8.0 for 20min at 96C (immerse slide rack in 250mL of antigen retrieval solution in covered beaker on hot plate, carefully monitoring temperature) Cool slides under running tap water for 10 minutes Use hydrophobic marking pen to mark an area around the tissue of interest Block slides in TBS/0.05% Tween / 0.3% BSA + 1ug/ml FC blocker #564220 BD ph + 25% FBS heat inactivated

Incubate in primary antibody (diluted in TBS/0.05% Tween / 0.3% BSA) overnight at 4 degrees C

Wash three times for five minutes in TBS 0.05% Tween-20 Wash once in TBS for five minutes

Incubate with Alexa 546-conjugated goat secondary antibody (1:100 dilution in TBS 0.05% Tween-20) for one hour at room temperature

Wash three times for five minutes in TBS 0.05% Tween-20 Wash once in TBS for five minutes

Dry area around sample with kimwipe Use Prolong Gold with Dapi (one drop) on sample and wet mount with glass cover slip Seal edges of cover slip with nail polish

Timing

Day one: One half day Day two: 2 hours

Consortium

(Re)Building a Kidney (RBK) Consortium