Does anyone have a good protocol for paraffin embedding adult rat brains? I don't have a tissue processor. But I can do it manually. thanks
In town or at your university there is for sure a pathology laboratory. Ask them for help and in most cases the technicians are willing to help you for embedding and cut the brains for you. Ask them how to fix the brains normally they also do neuropathology.
Dear Partha you cant do manually because out come of result will not be appropriate. You have to contact some pathology or university laboratory to take help.
Good luck
-First your tissue sample has to be fixed (post fixed eventually for a little time), if you intend to apply IHC. If for routine histology, you can postfix as longer as you want
-After, you have to dehydrate your sample by passing the sample twice in different graded baths of alcohol (50%, 70%, 90% and 100%)
- Finally, put the sample in a solvent bath twice (xylene or toluene for example)
- transfer then the sample in paraffin solution (60°C) for 1 min
- put it after in a mould with melted paraffin (at room temperature)
- Let processing the solidification. Wait 24 h and you can process sectioning
Let me know if you want more precisions
Dear Partha, knowing that this might be too sophisticated (since you lack specialized apparatuses and processors)....but nervertheless I would like to point you to the follwing (just to have an idea what you will be confronted in your future work:
Paraffin Processing of Tissue
Last update on July 15, 2012
http://protocolsonline.com/histology/sample-preparation/paraffin-processing-of-tissue/
You'll find also perhaps some comments (when the article has been accessed, set: CTRL&F,enter < brain > and RETURN]
Another general Paraffin embedding protocol, especially for IHC
IHC-PARAFFIN PROTOCOL (IHC-P) - by Abcam cf. http://www.abcam.com/ps/pdf/protocols/ihc_p.pdf
If you can't access that / these website(s), please let me know...I shall send you then a pdf of that webcontent.
Naturally there will be options or variations in protocol(s), depending on the final task (i.e., as has been said by Mohamed Najimi: only for histological / structural purpose, or also IHC etc.) and last but not least: every organ (human or from animal) usually has different claims for optimal processing. So you will find - when searching eg. Google (search phrase < e.g. RAT AND brain AND embedding in paraffin AND recipe AND OR method >) a lot of possible sources you - admittedly - have to go through some sources at least, I guess.
Best wishes, good luck, and regards.
Dear Partha,
The following steps are same for all tissues but the timings will vary depends on tissue and depends on the tissue of size.
hydration 4-5 hrs
dehydration with 50% ethanol 1 h
dehydration with 70% ethanol 1 h
dehydration with 90% ethanol 1h
Absolute alcohol I 1 h
Absolute alcohol II 1h
Absolute alcohol iii 45 min
chloroform 1-2 days
keep it your wax bath ready then go for xylene
Xylene i (its crucial step): time depends on colour change into gold colour
wax i 1h
wax ii 1 h
wax iii 45 min
Hope this may help
Good luck
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