Oct 23, 2023

Public workspaceYale Murine TMC - Tissue Preparation Protocol V.2

DOI
dx.doi.org/10.17504/protocols.io.kqdg3x8keg25/v2
  • Yun-Hee Youm1,
  • Yufeng Liu1,
  • Vishwa Deep Dixit1,
  • Rong Fan2,
  • Bo Tao2
  • 1Department of Pathology, Yale School of Medicine;
  • 2Department of Biomedical Engineering, Yale School of Engineering and Applied Science
Hester Doyle
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DOI: dx.doi.org/10.17504/protocols.io.kqdg3x8keg25/v2
Protocol Citation: Yun-Hee Youm, Yufeng Liu, Vishwa Deep Dixit, Rong Fan, Bo Tao 2023. Yale Murine TMC - Tissue Preparation Protocol . protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3x8keg25/v2Version created by Hester Doyle
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: October 23, 2023
Last Modified: October 23, 2023
Protocol Integer ID: 89748
Keywords: SenNet Yale Dixit TMC
Abstract
Protocol for embedding murine spleen, thymus and lymph node.
RNA Buffer (use before embedding tissue in OCT)
RNA Buffer (use before embedding tissue in OCT)
1x PBS-MC Recipe
  • All prepared in DNAse RNAase EDTA free deionized water
  • MgCl2 (1 mM, add 1 ml of 1 M MgCl2 stock solution into 1 L of 1x PBS)
  • CaCl2 (0.1 mM, add 2 ml of 0.1 M of CaCl2 stock soution into 1 L of 1x PBS)
  • 4% paraformaldehyde (PFA), optional
  • 1 X Protease inhibitors (Catalog # 04693132001, Roche-cOmplete , EDTA-free Protease Inhibitor Cocktail, Tablets provided in EASYpacks)
  • 1X Phosphatase inhibitor (Catalog # 4906837001; PHOSS-RO; Roche-PhosSTOP™ ; sufficient for 20-10 ml buffer preparations, suitable for tissue processing and immunoprecipitation)
  • RNAse inhibitors (Invitrogen SUPERase•In RNAse Inhibitor (20 U/μL ; Catalog # AM2696)

Filter sterilize with 0.22 μm syringe filter. Store at 4°C before use. Use within 2 weeks.
Critical
Incubate tissue on ice in the buffer for 15 minutes or during the collection.
Tissue Preparation/Embedding
Tissue Preparation/Embedding
Collect tissue in PBS on ice, wash if necessary
Dehydration/Fixation
Incubate the tissue in 20% sucrose/PBS solution or 4% paraformaldehyde and allow it to sink to the bottom of the vial at overnight at 4°C.
Embedding
Place the fresh tissue in the center of the mold (Tissue-Tek Cryomold #4566) filled with OCT (Tissue Plus OCT Compound, Fisher Scientific catalog # 23-730-571). Be careful to remove any air bubbles. Remove air bubbles in OCT bottle, then squeeze out.
Orient the OCT-embedded tissue into the desired position in the mold. Carefully squeeze out more OCT on top of the tissue, again avoiding bubbles. Squeeze enough OCT out until none of the tissue remains exposed.
Freeze the tissue block on dry ice (or in cryostat).
Wrap each block in aluminum foil or place into a small plastic bag. Store the tissue block at -80° C until ready for sectioning.