Nov 22, 2024

Public workspaceLung processing and fixation for cryosectioning

DOI
dx.doi.org/10.17504/protocols.io.q26g71p28gwz/v1
  • 1Washington University
Cecilia Escudero
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DOI: dx.doi.org/10.17504/protocols.io.q26g71p28gwz/v1
Protocol Citation: Carolina Lopez 2024. Lung processing and fixation for cryosectioning . protocols.io https://dx.doi.org/10.17504/protocols.io.q26g71p28gwz/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: May 28, 2024
Last Modified: November 22, 2024
Protocol Integer ID: 100731
Abstract
This protocol describes the fixation of lung tissue before cryopreservation. This protocol is required when the tissue will be used for staining intracellular proteins. 
Materials
ABC
Reagent IDVendor Catalog #
Fisher Healthcare Tissue-Plus O.C.T. CompoundFisher23-730-571
2-Methylbutane (Isopentane)Millipore-SigmaM32631-4L
PROTOCOL
PROTOCOL
7h
7h

Note
NOTE: Steps 1-7 can be done in a 12 well plate.


Perfuse and lavage the lungs with PBS
Inflate the lungs with Amount0.75-0.9 mL of 2% PFA/50% OCT and tie the trachea
a) Be careful not to overinflate.
b) Gently lift the inflated lung and remove it with the help of scissors.
Place the lungs in 4% PFA (can be done in a 12 well plate – Amount2-3 mL )
Incubate Duration01:00:00 at room temperature rocking the plate
a) This incubation can be done overnight, at Temperature4 °C .
1h
Decant PFA and rinse with PBS at least 4 hours
a) The PBS wash step can be done overnight, at Temperature4 °C .
Keep the tissue at room temperature for 30-60 minutes and transfer sequentially to:
a) 5% Sucrose, Duration00:45:00
b) 5% Sucrose: 20% Sucrose 2:1--------- Duration00:45:00
c) 5% Sucrose: 20% Sucrose 1:1--------- Duration00:45:00
d) 5% Sucrose: 20% Sucrose 1:2--------- Duration00:45:00
e) 20% Sucrose--- overnight at Temperature4 °C .
3h
Incubate the tissue in the infiltration solution for Duration02:00:00 at room temp.
a) INFILTRATION SOLUTION: Mix 40% Sucrose : OCT 1:1. Do not vortex the infiltration solution, as too much foam will form. Better swivel it.
b) Final concentrations of the infiltration solution: 50% OCT/ 20% Sucrose in PBS
2h
Prepare a Amount500 mL beaker with around Amount300 mL of Isopentane or 100% Ethanol. Fill a Styrofoam box with dry ice and shove the beaker inside, to create a dry ice bath. Cap the box until the freezing step.
Transfer tissues from the plate to labelled individual plastic cryomolds. Arrange the tissue as desired and add a drop of 100% OCT (sufficient amount to just cover the tissue) and incubate the cryomold + tissue at Temperature4 °C for Duration01:00:00 .

1h
Using tweezers, gently immerse just the bottom of cryomold in the dry ice bath (Beaker containing 100% ethanol or isopentane, surrounded by dry ice in a styrofoam box prepared in the step #8) and wait until the tissue piece is frozen / fixed to the mold;

Slowly add more 100% OCT on top of the frozen part, filling the cryomold up to 1 cm;

Finish freezing the entire cryomold, avoiding ethanol to get inside the mold and/or erasing the labels. If using Isopentane, you can fully immerse the mold and leave it for a while;

Transfer frozen molds to dry ice until the whole tissue is frozen

Cover in aluminum foil and put in a plastic bag or cryobox. Store at Temperature-80 °C .

Cut and stain as desired.
Protocol references
Adapted by Italo Castro