Jun 05, 2024
KAPP-Sen TMC: Tissue Section Preparation and H&E Staining
- Juliana Alcoforado Diniz1,
- Ramalakshmi Ramasamy1,
- 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, Ramalakshmi Ramasamy, Paul Robson, Elaine Bechtel 2024. KAPP-Sen TMC: Tissue Section Preparation and H&E Staining. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpzzkjlzp/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: June 05, 2024
Last Modified: June 05, 2024
Protocol Integer ID: 101261
Keywords: SenNet KAPP-Sen TMC, eosin stain, rnase zap wipe, further spatial transcriptomic analysis, performing further spatial transcriptomic analysis, jax histology core in bar harbor, jax histology core, rnase zap, spatial transcriptomic, tissue section preparation, following protocol glove, hematoxylin, clean glove, sen tmc, protocol glove, handling ffpe block, tissue
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Abstract
Before performing further spatial transcriptomic analysis, FFPE blocks were submitted to hematoxylin and eosin stain. All these procedures were performed by JAX Histology Core in Bar Harbor, ME where blocks were always kept at 4C. 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.