Protocol_Tissue and fluid collection following pancreatitis (Bronchoalveolar lavage (BAL),
Lung, serum):
Materials:
Microdissecting tweezers curved
Microdissecting tweezers straight
Kelly dissecting scissors (28-mm)
Catheter; Micro vascular clip 1-2mm (Roboz Item# RS-6472).
Sterile syringe (1 ml).
Reagent: 1 x PBS
25 ¾ G needle
Heparin
Tissue Casettes
4% Paraformaldehyde
Procedure –BAL and Lung Collection
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Using Kelly dissecting scissors make a small incision in the neck.
Using microdissecting tweezers remove the muscle layer surrounding the trachea, this will
expose the trachea.
Make a small semi-excision of the trachea to allow cannulation with a catheter (21-G lavage
tube) – take care not to cut through the trachea.
Once the catheter is in place, use a microvascular clip 1-2mm to hold the catheter in place. Load
a 1-ml syringe with 0.6 ml sterile 1xPBS, slowly withdraw the fluid by gentle suction immediately
after delivery –
Repeat this procedure 2 times in order to collect BAL (the amount of liquid withdrawn is less
than half the amount injected).
Expose the thoracic cavity and Inject the heart with 20mL of 1x PBS to wash the lung tissue of all
blood cells.
Infuse 1-2mL of 1x PBS through the trachea in order to have the lung tissue expand as much as
possible, once the lung is fully expanded the upper left lobe is quickly cut and placed in 4%
paraformaldehyde.
The rest of the lung tissue is placed in storage vials, flash frozen in liquid nitrogen and stored at -80C.
Procedure –Serum collection
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A laparotomy is performed and the guts are moved to the animal’s left side to reveal the inferior
vena cava located in the abdominal cavity.
A heparinized 1 mL syringe is used to cannulate the inferior vena cava
The blood is extracted slowly with necessary movement of the needle to facilitate flow of blood
into the syringe.
The blood is placed in a 1.5 ml eppendorf tube and kept on ice
Blood is spun at 10,000 x g for 5 min
Serum (i.e. supernatant) is collected and kept at -80C (for short term storage 4C is adequate)
Procedure-Pancreas collection
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Following blood collection, the pancreas is located (using the spleen as a marker for the tail)
A picture of the pancreas is taken as it appears inside the animal
Using fine forceps, the tail of the pancreas is held and the rest of the tissue is cut away from
neighboring small/large intestine.
The initial portion of the duodenum is located by looking towards the distal portion of the
stomach.
The head of the pancreas is located and the adjacent small intestine section is taken along with
the rest of the organ and spleen (this should result in one long piece of pancreas tissue flanked
by spleen at the tail and duodenum at the head)
If the pancreas is excessively bloody it is briefly washed in cold 1X PBS
The pancreas is laid out alongside a ruler and a second picture is taken
The head and duodenum are separated in one piece from the rest of the tissue and placed in a
tissue cassette. Remaining pancreas tissue is sectioned (as necessary) and flash frozen in liquid
nitrogen.
The cassette is placed in 4% paraformaldehyde
The cassettes are delivered the following day to the histology core in 4% paraformaldehyde.
The histology core embeds the tissue in paraffin, sections the tissue on glass slides and stains
with Hematoxylin and Eosin