Good evening everybody,
I am working on a model of acute heart failure and I am planning to analyse heat shock proteins (western blot) in the right and the left ventricle.
The mouse heart is very small (180 mg) and to divide the left ventricle from the free wall of the right ventricle has to be tricky.
Do you recommend to cut fresh (after the mouse sacrifice) or to freeze and cut it after that?
Thank you!
Hi Ruben.
In my experience it is always easiest to cut and prepare tissue at the time of sacrifice - that way orientation is also easiest to determine! I like to prepare my tubes for each downstream analysis step in advance of sacrifice so that when the time comes I can section the tissue and place in each tube as needed knowing I have the right piece in the right tube
Dear Ruben,
It is better to cut it immediately on ice after sacrifice foe each mice individually, immerse in liquid nitrogen and then keep in -80 C.
If you freeze the heart, it is going to be hard to cut it in two pristine equal weight pieces. I also recommend cutting the tissue right after the harvest, because you're going to get a better visual and physical control over the procedure. Both halves will be more or less even in weight. Besides, cutting the organ will eliminate a lot of blood from the chambers, so you will have less contamination of your protein sample. This is one variable that you have to worry about, because there are plenty of HSP proteins in platelets (Platelets. 2000 Feb;11(1):6-22) and possibly other blood cells. Ideally you would want to perfuse the heart to get rid of all blood, but then again you have to think what effects the shear stress will have on the expression of HSPs, as they, after all, are stress-inducible proteins. Even such seemingly innocent action as placing your tissue on ice must be standardized in time, because the duration of cold ischemia may also affect HSP levels (e.g. Hsp 70). The change may not be significant for a minute or two, so be quick. If chopping the heart into 2 pieces takes you only 15-30 seconds, I would not even place it directly on ice - a pre-chilled petri dish should suffice. Tricky procedure will remain "tricky" maybe for 2 or 3 rounds: afterwards your timing will significantly improve and your workflow will be reproducible and consistent.
Dear Edita,
this was what worried me: a procedure able to minimize effects on hsp. For my study I separate right ventricle, left ventricle and septum. This procedure takes me 3 minutes ( i can improve it). This time I used RNAlater solution after the dissection to "block" the proteins, then cut the heart, wash them in PBS, lysate with ice cold lysis buffer and sonicate. I'll have some results tomorrow!
Well, RNAlater is good if you are going to extract RNA and protein from your tissue simultaneously, but just in case, consider adding the broad protease and phosphatase (in case you'll end up looking at phosphorylated proteins) cocktail inhibitors in your lysis buffer prior to homogenization and/or sonication for 2 reasons: 1) the time may not be enough for RNAlater to permeate the tissue; 2) the ingredients of RNAlater (quaternary ammonium sulphates and cesium sulphate) may not inhibit all types of proteases. Anyway, good luck tomorrow!
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