Sep 01, 2023
KAPP-Sen TMC: Tissue Section Preparation and H&E Staining
- Juliana Alcoforado Diniz1,
- Paul Robson1,2,3,
- Elaine Bechtel4
- 1The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA;
- 2Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, Farmington, CT, USA;
- 3Institute for Systems Genomics, University of Connecticut, Farmington, CT, USA;
- 4The Jackson Laboratory, Bar Harbor, ME, USA
- Cellular Senescence Network (SenNet) Method Development Community
- KAPP-Sen TMC
Protocol Citation: Juliana Alcoforado Diniz, Paul Robson, Elaine Bechtel 2023. KAPP-Sen TMC: Tissue Section Preparation and H&E Staining. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr3653vmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 31, 2023
Last Modified: September 01, 2023
Protocol Integer ID: 85734
Keywords: SenNet KAPP-Sen TMC, rnase zap wipe, rnase free sectioning, rnase zap, rnase free sectioning for spatial transcriptomic, eosin stain, analysis of the whole pancrea, tissue section preparation, spatial transcriptomic, jax histology core, whole pancrea, genomic medicine, handling ffpe block, jax histology core in bar harbor, sen tmc, following protocol glove, joslin diabetes center
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Abstract
To perform imaging analysis of the whole pancreas the FFPE blocks were submitted to hematoxylin and eosin stain. Initially FFPE blocks were shipped from the Joslin Diabetes Center, University of Harvard Medical with ice packs to The Jackson Laboratory for Genomic Medicine (JAX) and kept at 4°C until submission to the JAX Histology Core in Bar Harbor, ME.
Upon arrival the blocks were kept at 4°C to undertake an RNase Free Sectioning for Spatial Transcriptomics. For the following protocol gloves cleaned with RNase Zap wipes must be worn during preparation for and completion of this protocol and whenever handling FFPE blocks intended for spatial transcriptomics. If gloves contact a surface not previously cleaned with RNase Zap during this protocol, clean gloves again.
Preparation of the Workspace
Prepare workspace according to https://dx.doi.org/10.17504/protocols.io.36wgq37polk5/v1
Section Collection
Note
Before beginning section collection, check project instructions for curl request. If curls are also requested, they will need to be collected immediately prior to section collection.
Blocks arrive wrapped in aluminum foil to protect from light and are stored in the dairy cooler at 4°C.
Wearing nitrile gloves cleaned with RNase Zap wipe, collect sections as follows:
Using a Histo-Quill Pen, hand label all slides with iLabs project number, block ID and stain if requested.
Retrieve blocks from 4°C dairy cooler and remove aluminum foil. Protect from light as much as possible during section collection.
Using an RNase Zap cleaned blade, face into block.
Using ice tray/block prepared per Part One, step 3 above, allow block to cool and soak if needed. If using ice block, add enough di water to cover ice so blocks won’t freeze to surface. Cover blocks to protect from light.
Using water bath, forceps and brush/applicator stick prepared per Part One, steps 2 and 4, section at 5um, discarding the first two sections before taking a ribbon. Collect sections on corresponding slides previously hand labeled in step 1.
After sections are collected, transfer unstained slides to rack prepared per Part One, step 5, and slides for H&E to Leica staining rack. Cover racks to protect slides from light.
Dip block face into molten paraffin to seal the tissue and allow to cool before rewrapping in aluminum foil and retuning to 4°C dairy cooler for storage.
Repeat steps 3 through 7 with remaining blocks, using RNase Zap wipe to clean blade, forceps and brush/applicator stick between each block.
Routine H&E Protocol: Leica AutoStainer XL
| A | B | C | D | E | |
| Step | Station | Reagent | Time, min:sec | Exact | |
| 1 | 1 | Xylene | 3:00 | No | |
| 2 | 2 | Xylene | 3:00 | No | |
| 3 | 3 | Xylene | 3:00 | No | |
| 4 | 4 | Ethanol, 100% | 2:00 | No | |
| 5 | 5 | Ethanol, 100% | 1:00 | No | |
| 6 | 6 | Ethanol, 95% | 1:00 | No | |
| 7 | 7 | Ethanol, 70% | 1:00 | No | |
| 8 | Wash 2 | Tap Water | 1:00 | No | |
| 9 | 9 | Mayer’s Hematoxylin | 4:00 | Yes | |
| 10 | Wash 4 | Tap Water | 2:00 | No | |
| 11 | 8 | Ammonium Hydroxide Water, 0.1% | 1:00 | No | |
| 12 | Wash 5 | Tap Water | 1:00 | No | |
| 13 | Wash 3 | Tap Water | 2:00 | No | |
| 14 | 10 | Ethanol, 70% | 1:00 | No | |
| 15 | 11 | Eosin | 2:00 | Yes | |
| 16 | 12 | Ethanol, 95% | 0:45 | Yes | |
| 17 | 13 | Ethanol, 95% | 0:30 | Yes | |
| 18 | 14 | Ethanol, 100% | 2:00 | No | |
| 19 | 15 | Ethanol, 100% | 2:00 | No | |
| 20 | 16 | Ethanol, 100% | 1:00 | No | |
| 21 | 17 | Xylene | 2:00 | No | |
| 22 | 18 | Xylene | 2:00 | No |
Slide Scan Submission
Slides will then be scanned by the JAX Light Microscopy Core in Bar Harbor, ME using Nanozoomer (Hamamatsu) HT2.0. Images will be acquired with brightfield scans at 40X.
Images can be accessed by NDP software or submitted to further imaging analysis at The Jackson Laboratory for Genomic Medicine, Farmington, CT. These images are also used to determine the Region of Interest for further Spatial Transcriptomics assays.