Hi Andrew:

Andrew Gray wrote:

>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.
>
There is no reason to fix at 4 degrees C, you just slow down the fixation.

>- 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.
>
Way too cold! This is probably the source of your problem. Try this method.

1. Chill some 2-methylbutane ('isopentane') with dry ice or by 
surrounding a metal beaker of 2-MB with liquid nitrogen.
2. Put the brain on a metal cryostat chuck ("object disk") with OCT.
3. Put the stem of the chuck into the chilled 2-MB. Freezing front will 
pass rapidly up the brain. If the chuck is "stemless" get a round, solid 
metal (brass, aluminum) bar of the appropriate diameter. Put one end in 
the chilled 2-MB, the chuck sits on the other end.
4. After the brain freezes, move it to the cryostat and wait 15 min for 
the temperature to stabilize

Alternative freezing for the low-budget lab:
Roll some heavy paper into a cylinder somewhat larger than the brain.
Wrap some dry ice in a paper towel, crush to powder with a hammer.
Place paper cylinder over brain, pour crushed dry ice into cylinder to 
freeze brain.

>- 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
>
Good luck!
Geoff

-- 
--
**********************************************
Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029 
mcauliff@umdnj.edu
**********************************************