Feb 02, 2023

Public workspaceOMS Atlas OCT Spatial Mapping V.4

DOI
dx.doi.org/10.17504/protocols.io.261ge677dl47/v4
  • 1Oregon Health & Science University;
  • 2Knight Comprehensive Cancer Institute, Oregon Health & Science University
Brett Johnson
Open access
DOI: dx.doi.org/10.17504/protocols.io.261ge677dl47/v4
Protocol Citation: Brett Johnson, Danielle Galipeau, George Thomas 2023. OMS Atlas OCT Spatial Mapping. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge677dl47/v4Version created by Brett Johnson
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: August 26, 2022
Last Modified: February 02, 2023
Protocol Integer ID: 69247
Abstract
This protocol describes the procedure by which the OMS Atlas serially sections an OCT block, prepares the resulting slides and samples, and then distributes the specimens for downstream analysis.
Materials
MATERIALS
ReagentSuperfrost Plus Microscope SlidesFischer ScientificCatalog #12-550-15
ReagentTanner Scientific 45° White Adhesive Slide with Beveled EdgeMercedes MedicalCatalog #TNR WHT45AD
Reagent1.0mm PEN membrane covered slides ; 100piecesZeissCatalog #415190-9041-000
Additional equipment:
  • UV lamp
  • Cryostat
  • Cryotubes
Before start
Transfer OCT blocks to OHSU Knight Histopathology Shared Resource (HSR) for sectioning and processing.
Preparation
Preparation
Verify the identity of the OCT block to be cut against written request for sectioning.
Remove OCT block from Temperature-80 °C freezer and acclimate to cryostat (Temperature-20 °C ) for minimum of Duration03:00:00 .

Label all slides and cryotubes with a unique BEMS ID and Part#, corresponding to the written request and OCT spatial map (below).
ABCDE
Part#DescriptionThicknessAssayRecipient
1Superfrost Plus slide5µmH&EOHSU, HSR
2Superfrost Plus slide5µmCyclic Immunofluorescence (Tumor Panel)HMS, Alyce Chen
3Superfrost Plus slide5µm (Set Cryostat at 12µm)Cyclic Immunofluorescence (Tumor Panel)HMS, Alyce Chen
4Cryotube7µmSingle Cell DNA SequencingMD Anderson, Nick Navin
5PEN membrane slide12µmTopographic Single Cell SequencingMD Anderson, Nick Navin
6PEN membrane slide12µm (Set Cryostat at 40µm)Topographic Single Cell SequencingMD Anderson, Nick Navin
7Cryotube40µm (2 sections)Single Cell DNA SequencingMD Anderson, Nick Navin
8Remainder of OCT blockNASingle Cell Indexing ATAC SequencingOHSU, Andrew Adey

Prepare PEN membrane slides by exposing close (~15-20cm) to a UV source for Duration00:15:00 .

Sectioning
Sectioning
Affix OCT block to cryostat chuck.
Orient and face block to get adequate amount of core.
Note: Avoid excessive facing to reduce tissue loss.
Set cryostat to 5 micron sections.
Note: All sections cut from here on should be sequential. The serial order, adjacency, and consistent orientation of the sections are all important factors. Please note any deviations from the protocol.
Cut first three sections at 5 microns (Part#1-3) and affix onto appropriately labeled slide according to OCT spatial map (step #3 above).
Change section thickness to 12 microns.
Cut one section (Part#4) and place in a cryotube.
Note: This is an intermediate section generated when the Cryostat is switching thicknesses. The actual thickness of this section should be about 7µm.
Cut two sections (Part#5, 6) and place on appropriate membrane slides.
Change section thickness to 40 microns.
Cut 2 sections (Part#7) and place both sections in a single cryotube.
Place all slides, both cryotubes, and remaining OCT block in Temperature-80 °C freezer.
Note: No slides are to be fixed under this protocol.

Processing
Processing
Perform hematoxylin and eosin (H&E) staining on slide labeled Part#1 (see OCT spatial map in step #3 above).
Deliver unstained slides (Part#2, 3, 5, 6), cryotubes (Part#4, 7), and remainder OCT block (Part#8) to BioLibrary for distribution.
Note: Keep samples frozen at all times. Store at Temperature-80 °C . Transfer/ship on dry ice.