Protocol Citation: katesama 2022. ALS Mouse Tissue Processing. protocols.io https://protocols.io/view/als-mouse-tissue-processing-ch9nt95e
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: In development
We are still developing and optimizing this protocol
Created: October 21, 2022
Last Modified: November 23, 2022
Protocol Integer ID: 71694
Abstract
This protocol describes how to prepare tissues and blood for downstream applications after the ALS mouse study.
Blood & Plasma
Blood & Plasma
Blood and Plasma Processing
After cardiac puncture and blood collection in K3-EDTA tubes, invert the tube a few times to ensure mixing with EDTA coating and keep it at RT until centrifugation within 4 hours of collection.
Spin whole blood at 1,600xg for 10 minutes at RT.
After centrifugation, pipette the plasma off the top. Be careful not to disturb the clot at the bottom of the tube or the buffy coat, and do not aspirate any of the clot or blood cells. Place the plasma into a second vial.
Plasma can be stored at -20°C for short-term storage (<3 months). Store at -80°C for long-term storage.
Brain
Brain
Extract and place the whole brain in 10mL 10% formalin (approx 1:10 tissue:fixative) and then place the tube on ice.
Spinal Cord
Spinal Cord
Dissect the lumbar region of the spinal cord (red box) with a scalpel. Place in 10 mL 10% formalin and put tube on ice OR in 10 mL 2.5% glutaraldehyde 0.1mol/l cacodylate buffer to fix for electron microscopy at RT.
Place the remaining spinal cord sections in a cryovial and flash-freeze in LN2.
Gastrocnemius Muscle
Gastrocnemius Muscle
Gently stretch both pieces of muscle for 10s by holding onto each side of the tissue with forceps and lightly pulling.
Place both pieces of the gastrocnemius muscle in 10% formalin (approx 1:10 tissue: fixative) and then place the tube on ice OR in 10 mL 2.5% glutaraldehyde 0.1mol/l cacodylate buffer to fix for electron microscopy and leave at RT(no need to flash-freeze any muscle).
Liver, Spleen and Kidneys
Liver, Spleen and Kidneys
Liver, Spleen, and Kidney Processing (for molecular biology and histology)
Section the spleen by cutting a transverse cross-section at the largest extension of the spleen using a scalpel
First, use tweezers to place the smaller section in 10 mL 10% formalin (approx 1:10 tissue:fixative) and then place the tube on ice. Then, place the second section in a cryovial and flash-freeze in LN2.
Section the liver by cutting a transverse cross-section through the left lateral lobe (1) using a scalpel.
First, use tweezers to place the section of the left lateral lobe in 10 mL 10% formalin (approx 1:10 tissue:fixative) and then place the tube on ice. Then, place the remaining liver section in a cryovial and flash-freeze in LN2.
Section the kidneys by making a longitudinal cut through each kidney using a scalpel.
First, use tweezers to place a section of each kidney in one tube of 10 mL 10% formalin (approx 1:10 tissue: fixative) and then place the tube on ice. Then, place the remaining sections in a cryovial and flash-freeze in LN2.
Colon
Colon
First, lay out the colon flat as pictured and discard the proximal section. Then, separate the mid and distal sections using scissors or a scalpel.
Using the back of a pair of forceps, gently push on the colon to remove the contents.
Cut the mid-section in half and place one half in 1mL RNAlater solution. Then, adjust the second half of the mid-section into a tube shape using closed forceps and place it in 1mL of 10% formalin. Store both tubes on ice.
Place the distal section in a cryovial and flash-freeze in LN2.
Fixation and Cryprotection
Fixation and Cryprotection
Fix formalin samples overnight at 4C. Then, replace 10% formalin with cold 30% sucrose in PBS.
Leave overnight at 4C, then embed or store in PBS at 4C until embedding.
Leave tissue overnight at 4C in sucrose solution, then embed or store in PBS at 4C until embedding.
RNA Sample Storage
RNA Sample Storage
Leave colon samples in RNAlater overnight at 4C to allow the solution to penetrate the tissue. Then, flash-freeze the samples in LN2 and transfer them to -80.
Cryovial Storage
Cryovial Storage
After returning to the lab and spinning down blood, transfer all samples in the -80 and log the samples, their position, and details of the sacrifice in the spreadsheet.