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Whole mount immunofluorescent analysis of human cortical kidney slices (Version 1.0) | ATLAS-D2K Center

PLEASE NOTE: ATLAS-D2K closed July 31, 2025 and this website is for reference purposes only.

Whole mount immunofluorescent analysis of human cortical kidney slices (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

Andrew McMahon; Nils Lindstrom

Keywords

[‘immunofluorescent’, ‘3D imaging’, ‘human renal cortex’, ‘nephrogenesis’, ‘kidney development’, ‘cortical slices’]

Subjects

[‘Developmental biology’, ‘Immunological techniques’, ‘Gene expression analysis’]

Release Date

2016-08-01

Abstract

Whole mount immunofluorescent analysis of human cortical kidney slices

Procedure

  1. Carefully decapsulate the fresh kidney tissue in 1xPBS. The capsule interferes with antibody access. Be careful to not disturb the underlying tissue.
  2. Place surgical gauze in 10cm Petri dish and fill the Petri dish with 1xPBS. Place the kidney on the wet surgical gauze. With a pair of blunt-ended forceps hold the kidney and with the other hand slice off approximately 3mm thick cortical slices – note, these are fragile.
  3. Fix kidney slices in 4% PFA in 1xPBS for 45 minutes at 4˚C without shaking. Wash out the PFA with several rounds of 1xPBS.
  4. Block the slices for 1hr. in 1xPBS with 2% SEA Block and 0.1% TritonX100 at 4°C with gentle movement (e.g. on a Nutator).
  5. Resuspend the primary antibody in blocking solution and incubate in primary antibody at 4°C with gentle movement for 48 hr.
  6. Wash samples for up to 8 hr. through several rounds of 1xPBS with 0.1% TritonX100.
  7. Resuspend the secondary antibodies in blocking solution and incubate the sample in secondary antibodies at 4°C with gentle movement for 48 hr.
  8. Repeat the washing steps and place in 1 µg/ml Hoechst 33342 for 2 hr. before a final wash in 1xPBS.
  9. Dehydrate the sample prior to clearing by passing the sample through a series of increasing concentrations of methanol (50%, 75%, 100%) 1 hr. for each.
  10. To clear specimens there are a number of options. For example, place the dehydrated tissue in a 50% Benzyl alcohol, Benzyl benzoate (BABB – note BABB is toxic) / 50% methanol solution for an additional hour.
  11. Replace with 100% BABB.
  12. Store sample at 4˚C in the dark until ready to image.
  13. Image as appropriate (e.g. on a confocal microscope).

Consortium

(Re)Building a Kidney (RBK) Consortium