Paraffin embadding protocol for mouse liver, spleen, thymus - (Jan/05/2006 )
I've just started with IHC and already got the problems with the cutting step. The preparation of the sample was done as following: piece of the spleen was fixed for 2h in PAF, after dehydrates in Ethanol (increasing concentration, each 20min), than stained in toluel and after placed in paraffin ON for tissue embadding. After solidifying on the next day of the sample, I've started to cut. First half of the sample was more or less OK, but when I wented deeper in the organ, I've got the problems: the sample started just to fall out of the paraffin and after that I was not able to place anything on the slide. Does somebody know, what could be the problem and do I need to change the time of fixation or dehydratation? Or may be somebody could provide me with the working protocol for mouse organs, I would like to do paraffin sections of the mouse liver, spleen and thymus.
Thank you very much in advance, Ksenia
This could be a multitude of problems. I suggest you get a text on basic histological techniques as it is too much to go into details but with a little background reading you soon would be able to troubleshoot this yourself.
It sounds like to me that your tissue is likely underfixed and/or underinfiltrated with wax (how thick are your pieces and how big in length and width??).
I strongly suggest that you also consider to use a core service if available or outsourcing your tissue to a histology service for paraffin embedding. If your lab does not routinely do paraffin embedding it can be tricky to do.
Please contact me at MaximinaMUA@yahoo.com and I will send you a protocol via email for some modifications to your protocol below but basically you probably need to do longer xylene incubations and definitely need to fix longer, you probably also need to prepare your tissue differently and your blocks before cutting.
I had a similar problem, and learnt the hard way that embedding is extreamly sensitive, and if not done properly if will also give you problems with retrieval and antibody staining.
I would also suggest that if you can, send your samples to a histological service for fixing, or see if you can learn from them, sometimes you need to see how its done to pick up the little tricks they use.
All the best!
i also did some sections for IHC with various of tissues of mouse including liver, spleen, ... i have no problem in doing it, this is how i did
fix with 4% PFA o/n, PBS 15min, 50% EtOH/saline o/n, 70% Etoh, then 100% etoh o/n. (time may reduced depends on tissue size) then toluene until tissue become clear (3x 15min)
add wax in 65oC 2 hr, change 1 time and add minmum of wax to tissue and place RT for o/n. next day add wax in 65oC for 1 -2 hr. then embed
i usually section them in 5u thick and make sure u move ur section down the blade slowly to prevent it to breakdown