[Histonet] Re: Parrafin tissues crumbles

[Galina Deyneko]

Hi Michael.
1. Fixation : 48 hours at room T is a good time to fix a big rat heart. You can transfer at 4C after 24/48 hours, otherwise cold T from the beginning prevents good penetration and fixation.
2.. process the hearts and muscles separate from other tissues
3. use only fresh reagents in you processing machine. I always rotate reagents before process the hearts  and this really works for me.2 hours in each Ethanols is OK in my opinion but according me experience it depend on processing center .I think that you use xylene substitute and I can recommend to switch back to real Xylene's. the clearing in Xylene is very short -30 Minutes?, especially after long dehydration. It would be better if you use 1.30 or 2 hours on each. Time in paraffin is OK, but I prefer 3 baths of paraffin, 1.30 or 2 hours in each. In the processing center use paraffin called infiltration medium i use from surgipath # 01400, for embedding i highly recommend Shandon Precision Cut paraffin from Thermo Shandon# B1002490.
Do not freeze paraffin blocks this make the paraffin and tissues very fragile. You can cool the blocks in refrigerator or on the top of cold plate.
Trim the paraffin block and place in the DI water for soaking for 5-6 minutes, do not keep to long. Use only DI water in the floating bath. Moisten the cutting surface of the block with your finger during sectioning, this is help to get more smooth sections. 
My English is not perfect but I hope you understand my tips.please contact with me if you have additional questions.
Galina Deyneko
Novartis, Cambridge, MA
617-871-7613
galinadeyneko <@t> yahoo.com
 

Message: 8
Date: Tue, 9 Nov 2010 14:56:49 -0800
From: "Michael Mashore" <mmashore <@t> vapop.ucsd.edu>
Subject: [Histonet] Paraffin Tissue Crumbles
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <652586E049884D8491A0E70448595D40 <@t> vandeberg>
Content-Type: text/plain;    charset="iso-8859-1"

Hello Histonet Users,



I have just started using paraffin and am having many difficulties. Most of
the time my tissue crumbles when sectioning. I have no real experience in
paraffin histology and have been given the task of becoming proficient by
myself, so I am hoping for feedback as to why my tissue keeps crumbling. The
tissue in question has been:  skeletal muscle, cardiac muscle, liver, and
brain (all from rat).



The tissue was fixed in 10% neutral buffered formalin for 7 days at 4°C and
then transferred to an automated tissue processor, with the following
schedule:



2 hours 70% dehydration alcohol 

2 hours 80% dehydration alcohol

2 hours 95% dehydration alcohol

2.5 hours 95% dehydration alcohol

2 hours 100% dehydration alcohol

2 hours 100% dehydration alcohol

2 hours 100% dehydration alcohol

.5 hour Hemo-De

.5 hour Hemo-De

.5 hour Hemo-De

1 hour paraffin

4 hours paraffin



They were infiltrated for 1 hour without vacuum then embedded.

The blocks were stored in the freezer before cutting.

The knife angle was 5°. 

Sections were 5µm thick.



I would appreciate any feedback whatsoever. 



Thank you very much.



Michael



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