Back to Manual - Chemical Fixation

 

This describes the protocol for diffusion of cryoprotective agent (CPA) into a brain that is already fixed with formalin and glutaraldehyde.

 

Cryoprotection Ramp

The ramp for EG is as follows 

%CPA	Brix	%Target
0%	7.9	0%
8%	13.1	12%
16%		25%
32%		49%
65%	43.9	100%

 

The ramp for EG/DMSO mix is as follows 

%CPA	Brix	%Target
0%	3.7	0%
8%	10.3	12%
16%	16.8	25%
32%	29.2	49%
65%	47.2	100%

 

The concentrations listed in the table should be thought of as defining an ideal smooth exponential curve like this:

 

 

But It's not practical to increase concentration continually, so steps are used that track the curve as closely as possible.  Brix is used on the vertical axis to avoid frequent conversions.  The ramp then looks more like this:

 

 

PBS

Phosphate Buffered Saline is a component of the mixture.  The current product is a 98.9 g packet of powder designed to make 10 liters.  We use the powder to avoid having to store solution and also to allow buffering of nonaqueous solutions.  The ratio is 9.89 mg/mL. 

 

Steps

All steps have now been moved to worksheets.  A new worksheet is used for each step of a ramp.  It contains all instructions as well as boxes to record measurements and notes.