4 replies to this topic

[peak99]

We have paraffin embedded blocks of rat brain tissues for which we have been able to get most of our antibody/IHC protocols working on 12um sections.

We found that some of our blocks were not sectioning, (the tissue curled while the wax cut) but humidifying the section for an hour @ 37C made sectioning much easier.  

These sections do not unwrkinkle completely in the water bath and do not stain (for antibody detection) under our protocols that have worked thus far.  These sections also have a likely tendency to loosen and partially come off the slides following HIER in EDTA( in the microwave), although not always and is inconsistent and the counterstain works fine.

I am looking for opinions on what may be the problem with these sections/blocks...
1) they have a distinct color in the block (darker in general in these problematic blocks) what could cause this? fixation or embedding or something else?
2) requires humidifying to section.
3) negative for detection of numerous ab markers with previously validated protocols (with other tissues from the same study). is this due to the same problem? we don't know. we are going to try microwaving (HIER) for a shorter time and see if that helps.

What is different about the blocks not working:
1) whole brain  (not working) versus cerebellum blocks (working)
2) embedded by different people (although on the same automated machine second batch are the ones not working). Thus the suspicions that the embedding is the root cause.

thus any opinion on factors that may cause this/solutions, etc. are most appreciated for consideration.

thanks.

[bob1]

Not an expert by any means, but from your post I came to the same conclusion as you that the embedding or perhaps the fixation process was a problem.  Darker wax could indicate contamination with one of the many solvents used in pre-processing, probably xylene, which would make the wax softer. It could also be that the wax was too hot and destroyed the epitopes.

[peak99]

This is on hold at the moment in our lab, but I agree i think it is the embedding or maybe fixation.

The colour of the wax in the block is not evident but the tissue is a darker colour than our "good" samples within the block. And the only thing different is the time and operator for embedding...so it is probably the root cause.

Now, do you think there is anything we can try to get these problem tissues to stain? *sigh*

[bob1]

You can try more aggressive epitope retrieval, but it may not work.

[stevenhisto]

Maybe you could try bouin fixation in combination with a longer incubation time or predigesting with enzymes.