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Digoxygenin-labeled in situ Hybridization protocol on Frozen Kidney Sections (Version 1.0) | ATLAS-D2K Center

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

Digoxygenin-labeled in situ Hybridization 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

Keywords

[‘in situ hybridization’]

Subjects

[‘Molecular biology’]

Release Date

2020-08-20

Abstract

mRNA In Situ Hybridization protocol on 10um Frozen Mouse Kidney Sections

Reagents

Paraformaldehyde (Sigma, P-6148) 1x PBS pH 7.4 30% Sucrose/PBS (solution must be heated and agitated for sucrose to go into solution Triethanolamine (Sigma, 90279) Acedic anhydride (Sigma, A6404) HCl Hyb buffer 50% formamide (Fisher, BP227 100) 5x SSC pH 4.5 (use citric acid to adjust pH) 50ug/mL yeast tRNA (Gibco, 15401-011) 1% SDS 50ug/mL heparin (Sigma, H8514) Proteinase K, pK (Roche 161519) Tween-20 (Sigma, P1379) Blocking reagent (Boehringer Mannheim, 1096 176) FBS Anti-Dig-AP (Roche, 1093 274) Levamisole (Sigma, L9756) BM Purple (Roche, 1442 074)

Procedure

Tissue Preparation

  1. Isolate kidneys
  2. Fix in 4% PFA/PBS at 4 degree overnight
  3. Rinse 3X in 1x PBS pH 7.4 for 5 min each
  4. Treat kidneys in 30% sucrose/PBS overnight
  5. OCT wash
  6. Freeze in OCT
  7. Store kidneys at -80C
  8. Section kidneys into 10-12 um sections
  9. Store slides in -80C

Make anti-sense RNA probe

directions provided in another protocol

in situ Hybridization Protocol

Day1 ~3.5 hours

  1. Preparation For cryosections, take slides out of -80C, let dry at room temperature for ~10minutes Prepare solutions i. PBS (ph to 7.4, with calcium and magnesium, MQ) ii. Fresh 4% PFA in PBS iii. PK in PBS (30ul of 10mg/ml pK in 20mL 1x PBS pH7.4 per every five slides) iv. Fresh Acetylation buffer in coplin jar on stir plate with a stir bar (200mL miliQ water, 2.66mL triethanolamine, 350uL HCl) Turn on 68C incubator Locate probe.

  2. Fixation Wash slides in PBS 3x5 minutes Fix sections in 4% PFA for 10 minutes (save this PFA for step 4) Wash slides in PBS 3x3 minutes

  3. Proteinase K treatment pK treatement (20ml per slide mailer) i. time is important here!! Tissue determines time ii. E18.5 10um kidney section – 10minutes rinse in PBS briefly ~3 minutes

  4. Fixation Fix sections in 4% PFA for 5 minutes (use the PFA from step 2) Wash slides in PBS 3x3 minutes Put hybridization buffer in 68C for step 6

  5. Acetylation Add slides to acetylation buffer in coplin jar (step 1iv) Add 750 ul acetic anhydride Let stir for 10min at room temp Wash slides in PBS 3x5minutes

  6. Prehybridization – in humidified chamber; lots of water Remove excess PBS by blotting with a kimwipe Take out hybridization buffer to room temperature for 3 minutes Add 250ul hyb buffer to each slide, place parafilm on each slide Let sit at room temperature for 30min-1hour Warm hyb buffer in 55C Heat probe in hyb buffer at 80C for 30min Dilute probe if necessary i. 1x probe – add 250uL directly to slide ii. 2x probe – add 125uL probe + 125 uL hyb buffer (warm) per slide iii. 10x probe – 25uL probe + 225uL hyb buffer (warm) per slide

7.Hybridization Add 250ul probe/slide Add parafilm to each slide Incubate in 68C incubator overnight

Day 2: ~2.5 hours

  1. Preparation Turn on incubator (72C) Warm 50ml conical of 5x and 0.2x SSC

  2. In situ hybridization – antibody Immerse eslides in 5x SSC warmed to 72C for 5 minutes to encourage parafilm to separate from slide Wash slides in 0.2x SSC at 72C for 30minutes, twice (incubator) Rinse slides in 0.2x SSC at room temperature x 5minutes on a nutator While washing, make NTT (will use this for 2 days, make fresh for each set of in situs) 20mL 1M Tris pH 7.5, 6mL 5M NaCl, 1mL 20% Tween 20, adjust volume to 200mL using miliQ water). Transfer slide to mailer with NTT (few minutes) Incubate slides in humidified chamber with 250ul block (5%FBS + 2% blocking reagent in NTT) for 1-2 hours at room temperature Blocking solution: 2% blocking reagent (Boehringer Mannheim 1096 176) in NTT. Solution must be heated and agitated for blocking reagent to go into solution Prepare antibody solution (1:4000 dilution of Ab [anti-digoxygenin-AP] in blocking buffer) i. Remove parafilm from slides, blot excess blocking solution ii. Add 250ul ab dilution per slide, cover with parafilm iii. Incubate 4C overnight

Day 3: ~2 hours 10.In situ hybridization – BM purple AP substrate Immerse slides in NTT to separate parafilm from slides Rinse slides in NTT at room temperature on nutator for 3x30min Rinse slides in fresh TTML for 3x5minutes (6mL 5M NaCl, 10mL 2M Tris pH 9.5, 1mL 20% Tween, 10mL 1M MgCl2, 400uL 1M levamisole, adjust volume to 200mL using miliQ water) Immerse slides in mailer wrapped in foil with BM purple that has been prewarmed to 37C Keep at 37C or room temperature monitoring closely as strong probes have a tendency to overstain quickly and background appears sooner and stronger. Check for signal

  1. Mounting slides Once developed rinse in PBS for 3x5minutes Fix slides in 4% PFA at room temperature for 1-2 hours or fix overnight at 4C Rinse slides in PBS for 3x5minutes Blot off excess liquid/dry well Mount with glycerol avoiding bubbles (Warm in water bath to 56-60C prior to use and between slides)

Consortium

(Re)Building a Kidney (RBK) Consortium