Dear Histonetters,

I have had so many requests for this method, pardon shortening  name to
GLUOX, got tired of writing it out.  Please give credit to Barb Wright who
shared her fixation protocol with me, some of you may already use
acetone/alcohol.  Dr. Craig Pow at Vector Labs tech services provided me
with the method, references several years ago.  

I included some hints on things I ran into, one lady a couple of weeks ago,
sorry I don't have her name - also uses this method and passed on the
microwave warming of the blocking solution.

Please remember that you may get some poor morphology on occasion, I think
my waterbath temp may flucuate more than I would like hence funky cateye
nuclei, spidery tissue - I just repeat a run.  No big deal, that's research
and my tissues.  

Thanks to all who provided the info on this method, I am in your debt!



>glucose Sigma G5250   0.180 g
>glucose oxidase Sigma G 6641  0.005g
>sodium azide  0.0065 g
>50 ml PBS sigma 1000-3 or DPBS from Sigma
>
>Add glucose oxidase to prewarmed 37C PBS.  I dissolve the glucose in the
>buffer, then add enzyme just before I put the slides into a 37C mixture.
>This temp is critical, do not go higher, it will eat sections.
>
>I make up a stock buffer of DPBS containing the correct concentration of
>sodium azide -  too nasty to keep weighing out, make up 5 liters or
>more at a time.  It does not get growth with the azide, and Sigma DPBS is
>great, the one without Ca and Mg.  weigh out 10 g in a liter of MilQ water,
>pH is always at 7.4.

Method:
Incubate 1 hour, remove and rinse gently with DPBS X 3

Fixation protocol for frozen sections is
>
>cut and air dry overnight, fix in acetone 75 ml/100% ethanol 25 mls for 5
>min at RT.  Go directly to a PBS rinse after fixation, start staining.
>
>IF you use just acetone, the sections often get a spiderweb effect, nuclei
>look funky.  Morphology changed to the better with this new fixation
protocol, plus I
>get wonderfully clean slides.
>
>I work with 100 mls of buffer, so double the contents and uvsgcI preweigh
>vials of 10 ug glucose oxidase, freeze down at -27C, then I just rinse the
>enzyme with prewarmed buffer, stir well but not long in prewarmed
>buffer/glucose, 
>I also prewarm my incubation coplin jar or staining dish, add prewarmed
>GLUOX to that.
>
>I use MW to prewarm my buffer/glucose, calibrate for your volume.  Do not
>overheat or you will have to cool to 37C before adding enzyme.
>
>Ref:  Andrew SM and Jasani B Histochem J.  1987, 19:426-430  
>
>      Hsu H-M, Yan K, Jaffee ES Am J Path 188:209-217, 1984 
>
>YOu can try acetone fixation but it is not so great for morphology with
>GLUOX, I am impressed with my clean results.  You will get some funky
>sections, depending on lots of conditions of tissue, disease, etc and if
>you don't have chemicals in right conc for GLUOX, plus temp flucuations.
>Have a good water bath, I do not use an incubator.
>
>
Gayle Callis
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
406 994-4705
406 994-4303