Paraffin sectioning for in situ

Henk Veldman H.Veldman-2 at neuro.azu.nl
Wed Jul 11 02:52:34 EST 2001


Vasundhara Kandachar schreef:

> Hi all!
>
> We are trying to prepare 10 micron sections of spleen,
> thymus and lymph nodes from 6-8 week old mouse for in
> situ hybridization (to mRNA).
>
> We have encountered two major (related?) problems:
>
> First, we've found long rectangular holes in the
> sections, as if a piece popped out while sectioning.
>
> Second, sometimes the entire section breaks into
> shards leaving only the parafin ribbon without tissue.
>  We have been told that perhaps these
> sectioning difficulties are due to dehydration or
> parafin infiltration problems.
>
> Our procedure (below) has been taken from established
> protocols for sectioning animal tissue.
> We process the tissue as follows:
> Fixation: 4% paraformaldehyde in PBS overnight (RT).
> Wash:  0.1 M Sodium phosphate buffer at 4 degrees C
> overnight.
> Dehydration:  5 minutes in each of six different
> concentrations of ethanol at RT (increasing from 10% ,
> 25%, 50%, 70%, 95%, and 100%).
> Replaced ethanol:  Used 50% xylene in 100% ethanol,
> then 100% xylene (both for 5 minutes at RT.  Paraffin
> Infiltration: added 2 pellets of parafin every 10
> minutes at 60 degrees C for 8 hours and then resumed
> adding pellets the next day for another 8 hours.
> Evaporate the xylene:  Left the vials without caps at
> 60 degrees C overnight.
> Replace with 100% parafin:  twice per day for 10 days.
> Pour parafin with tissue into molds and left 4 degrees
> overnight.
> Cutting sections:  Used hand operated microtome with
> freshly sharpened steel knife to cut 10 micron
> sections from blocks that have been trimmed and
> cooled on ice.
>
> Please help!  What are we doing wrong???
> (the person cutting the sections is a very experienced
> plant histologist who uses the same microtome
> successfully for preparations of thin sections for
> TEM).
>
> Thanks in advance.
> David.

It's quite some time ago that a prepared paraffin section. If a cryostat
is available I would go for cryo-sections for immunohistochemistry or
ISH.

However, if you want to stick to paraffin:
It sounds as if your tissue is not fully dehydrated and therefore it
cannot be infiltrated very well with paraffin, and indeed dehydration
times with alcohol are rather short. Moreover, ethanol tends to absorb
water, it is usually not better than 96 %. Either make sure your final
ethanol is really nearly absolute, or (more easy) use isopropyl alcohol
instead. If the tissue does not become fully translucent ("cleared") in
xylene it was not fully dehydrated. Also, since xylene tends to harden
the tissue, toluene or chloroform would be a better choice (remember:
chloroform will not make the tissue "clear"). And leaving tissue in
molten paraffin at 60 deg for 10 days also may make it brittle.

A typical traditional scheme (adapted for a normal 8 hour working day)
for tissue blocks about 3-5 mm thick would be:
1 - Fixation in buffered formalin
2 - Formalin/alcohol 70%: overnight
3 - Alcohol 70 %: 2 hours
4 - Alcohol 90 %: 2 hours
5 - Alcohol absolute I: 1 hour
6 - Alcohol absolute II: 1 hour
7 - Alcohol absolute III: 1 hour
8 - Toluene I: 1 hour
9 - Toluene II: overnight
10 - Paraffin I: 1 hour @ 60 deg
11 - Paraffin II: 1 hour @ 60 deg
12 - Paraffin III: 1 hour @ 60 deg
13 - Embed in fresh paraffin, making sure the paraffin around the block
is still fully molten when it is placed in the mold ( to avoid seams in
the block).
Larger blocks need to be processed slower.

I am a bit surprised that you are using the same microtome to cut
EM-sections (presumably ultrathin, with a glass or diamond knife) and
paraffin sections (10 micron with a steel knife). Are you sure the knife
angle and the cutting speed are OK?

Hope this helps,

Henk
--
H. Veldman
Laboratory for Experimental Neurology (NMZ)
University Medical Center Utrecht (AZU)





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