Liver Tissue Crosslinking

Revised 20 May 2007 -- Odom laboratory @ CRUK CRI

Note: We will FedEx you the correct amount of Solutions A, B, C, D in advance.

Process a maximum of 5mls of minced tissue in a 50ml starstedt tube.

Step 1: Mix Solution A with Solution B.

Add 1 volume of Solution B (caution: contains formaldehyde) into 9 volumes of Solution A for a 1:10 dilution.
For example: for 4 mouse livers you would mix 20 mls solution B with 180 mls Solution A.
Mix the right amount of solution A+B no more than 5 minutes before the procedure.
Anesthetize animal terminally, and begin procedure after complete non responsiveness.

Step 2: Flush liver and quickly flash freeze small portion of tissue.

If possible, flush liver with Solution D (optionally adding heparin if you have it on hand) to remove blood (this step is preferred, but optional).
Remove approximately 4, 0.5mm chunks of tissue and flash freeze in a well-labeled eppendorf tube (dry ice or liquid nitrogen if available).
This will be used for RNA isolation so avoid cross contamination between samples if possible.

Step 3: Continue to mince liver into Solution A+B.

Mince/slice liver into pieces thinner than 3 mm with razor blade or scalpel.
Place tissue in a 50ml tube and immediately add Solution A+B. No more than about 5-6 ml of tissue should be added to a 50 ml tube.
It is important to get the minced tissue into Solution A+B as quickly as possible.

Step 4: Incubate 20 minutes.

With occasional gentle shaking/stirring, let the tissues sit in Solution A+B for exactly 20 minutes at room temperature.

Step 5: Rinse with Solution C.

Drain off the Solution A+B by pouring off the solution through the metal collander provided.
- The liver should remain in the 50 ml tube, but if it doesn’t the colander will catch it and you can place any lost bits back into the 50 ml tube.
Cover the minced liver with 50 ml of solution C. Let sit for exactly 10 minutes with occasional gentle shaking/stirring.

Step 6: Rinse with Solution D.

Drain the Solution C off as above, rinse for 1-2 minutes with Solution D, drain, and freeze on dry ice or in LN2 in well-labelled tubes.
On the tubes, please have the animal tracking number, and date of liver processing.

Step 7: Freeze and ship!

Plastics and leftover solutions can be disposed of as desired. Please ship us the tissues on dry ice.

Solution contents

Solution A:

  0.1 M     NaCl 

  1.0 mM    EDTA

  0.5 mM    EGTA 

  50 mM     Hepes (pH 8)

Solution B:

  10% formaldehyde in distilled H2O

Solution C:

  2.5 M glycine (187.5 g/L) in distilled H2O

Solution D:

 1 x PBS

Please ship to the address below. If you have any questions at all please do not hesitate to call or email.

Duncan Odom
Cancer Research UK
Cambridge Research Institute
Robinson Way
Cambridge
CB2 0RE

Telephone: 01223-404500
FAX: 01223-403548