Hi histo people!

This may have been covered previously, but without having seen images 
I'm not certain.

I'm attempting fluorescent IHC and having problems with widespread 
microscopic cracks in my rat brain sections.   A typical image can be 
viewed at 
http://www.geocities.com/its_a_knockout/secondaryLMol_6se.jpg .   (Red 
fluorescence image, Texas Red secondary Ab, no primary Ab, hippocampus 
x10, camera exposure increased to show features).

Previously, there was a problem with the 'Swiss Cheese' artifact owing 
to slow freezing.   This problem has now been solved.   I suspect that 
the cooling may now be too rapid.   The pattern of cracks appears 
similar in adjacent sections.

Briefly, my method is as follows:

- Anaesthetise (Amytal) & transcardially perfuse rat with 0.1M PBS 
followed by 4% formaldehyde.

- Remove brain and postfix in 4% formaldehyde/10% sucrose 4degC O/N.

- Cryoprotect in 30% sucrose at 4degC till brain sinks (2-3 days), 
often longer.

- Dissect brain along saggital plane to obtain four equal-width chunks
(reduces incidence of macroscopic tissue shattering).

- Freeze rapidly by holding in weighing boat over liquid nitrogen.

- Section (saggital) 50u (thick!) at -20degC in a cryostat (we don't 
have a freezing microtome in our lab).

- Place sections in PBS (room temp) and process as free-floating 
sections, maipulating using a fine paintbrush.

- Mount on glass slides and coverslip with Vectashield mounting medium.


Thank you all for your time; the Histonet has been an absolute life 
saver for me in my PhD studies to date.

Best wishes,

Andrew Gray
PhD Student 
Monash University
Melbourne Australia