Paraffin Embedding

Hi all,
I have a muscle tissue which I want to embed in Paraffin.
I am going to do it like follows:
1- Over night in melted paraffin
2- 1Hr. Melted paraffin
3- 1 Hr. Melted paraffin.

The question is what temp. the paraffin should be , 58deg Cel is the melting temp.
Some protocol say to do it in 60 other say 65 deg Cel?

Any experience with muscle tissue paraffin embedding?
Guy

yes 60 degree C is fine, i had done lots of time with this and it works perfectly. higher than this temp. may cause hardness of tissue and cause dust formation during sectioning.

regards

Thanks for your response,

I had dust formation couple of times and it is also could arise from wrong dehydration.

I have another question to you,

When I am cutting the muscle tissue many times I some how lose the connecting tissue. So when staining it in Masson Trichrome I can see just the muscle bundles and some times the neuro vascular bundle. Do you have any ideas why I am getting this?

yes improper dehydration may cause dust formation during sectioning.

Regarding loss of CT during sectioning, this may cause due to improper paraffine embeding. You may do one thing that after processing of tissue, keep it in melted paraffine before making block (embeding), when you starts block making remove tissue from melted parraffine and make block immideatly which enhance the proper block making and make shoure that blade / knife is sharpe enough during section cutting. this may solve your problem.

regards.

as far as i think its not good to keep the tissue overnight at melting point as it can lead to protein destruction , and the temp should be 2 to 4 degree more than the MP of the wax you are using

yes overnight keeping in paraffine is not necessory. but the temperature of the paraffine should around 60 degree C, as their are product of praffine which have higher malting point and if you keep tissue in that may cause protein denaturation.

regards.

Thanks all,

I have lowred the temp. to 59deg cel.

I will do the paraffinisation 3 times 60 min each.

Another Question?

After dehydration last step  Xylene, can I leave it over night at RT out side the Xylene on the bench before putting it in the melted paraffin?

Guy

 after dehydration you have to put it in xylene and later on in parraffin , keeping it in xylene overnight will make your tissue brittle

It is not desirable to keep tissue out side, rather you can keep tissue in melted parraffine after xyline stape and let that parraffine solidify, in this you can keep it over night and very next day you can again start the process from parraffine - I, II, & III.

So stapes are

Xyline

keep tissue in melted Paraffine

Allow to solidify and keep it in for over night.

Next day start process - melted parraffine I, II, & III.

Regards.

When processing any tissue the temp of the paraffin should not exceed 60C.

It sounds like you are processing by hand and not on an automated processor.

First. Fixation. Fixation. Fixation. How big is your specimen? Unless larger than 1cm cubed, overnight in PFA or formalin should be fine.

Proceesing should be 70% EtOH , 2 changes 95% EtOH, 3 changes 100% EtOH, 2-3 changes xylene  30-45 mins each

3 paraffins 45mins to 1 hour.

If you cannot process in one day, the tissue should be left in the 70 or 95% EtOH. NEVER, ever leave the tissue out of a solution to dry!!!!!!!!!!!!!

Thanks Excalibur,

The sections are 0.5X0.5X0.5mm.

10% NBF for 5 days ( I know that 24 hr is OK)

The exchanges are done in 70% then 80% then 95% ( I tried each for 30 up to 60min)

100% I have used 95% Ethol + 5% meth, or 95% Ethol + 5% Isopro. 3 changes 1hr each

Xylene 2 Changes 30min up to 60min.

Paraffin below 60deg cel 3 changes two ways either first change Over night and then the other 2 1hr each, or all the three changes all together 2hr (40min each).

After paraffin I embeded the tissue.

Stii none of the above resulted in good sectioning. The tissue crumbles turns to dust.

I do not have any Idea what to do?

Thanks for helping all of you.

This is what Scientist Solutions is all about , people from different contintents helpping each other.

Guy

If you are using 95% EtOH and then adding 5% MeOH or Iso, the 95% still contained 5% water, which is probably your problem. There should be no water content at all in your 100% alcohol to process.

Are you using a 95% alcohol (5% water) or reagent alcohol which contains 95% EtOH and 5% other alcohols and no water?

Is the xylene turning a cloudy white when you place the tissue in it from the alcohol? If it is, there is water in your alcohol.

Hi. Platelet Pete here. I have been having some of the same issues with crumbling of tissues. In my case, intestine, lung and brain are okay with my processing method. I have problems with kidney, liver and spleen. I thought the issue was the tissue composition, that highly vascularized. This happened regardless if I used fresh alchols and xylene subsititute or xylene. We are using the same protocol as our histology core and they don't appear to have this problem. I thought it might be the xylene subsitute but didn't consider the paraffin temperature. I hope this helps.

I will subscribe to this forum.

Dear Sir

as i am reading this discussion one thing is sure that your tissue was left in 10%NBF for 5 days thats a too much of fixation, as you have not mentioned the thickness of your tissue before fixation, other thing is there are chances of removal of too much of bound water being released from the tissue  during dehydration as the dehdration has to remove only the free water molecules , the possible remedy for this may be keep your block before sectioning on ice or at 4 degree and keep moistening the exposed part of the tissue during cutting as it may compensate a bit for your excess dehdration if it had happened, rest is all irreversible you have try at new tissue with fresh protocol keeping the points raised in discussion at handy.....we will continue the discussion as it will cetainly expose the culprit...............................

You cannot overfix. Once the crosslinks are formed, the tissue can not undergo any more changes. IMHO, good fixation is the most important step.

As to your tissue crumbling during sectioning, this is caused by improper processing. The most likely culprit is water in the last alcohols. I have 30 years experience in histology and this is my opinion without actually seeing your specimens.