RE: alk phos stain in culture plates

<< Previous Message | Next Message >>
Content-Type:text/plain; charset="iso-8859-1"

There is a real simple enzyme method you can do, you get the reagents from
Sigma, I will list them here.  I have not done this with culter cells, but I
have done it with frozen sections and cyto spins and smears, it is very
strong and messy with unprocessed cells.  In frozens, the enzyme activity is
all over the place and hard to wash from the background, so I suggest you
react for short times and rinse really well.
For GMA processed bone sections:
Reagents:  Maphthol AS-MX Phosphate Alkaline Phosphate solution, 0.25%
(Sigma Cat. NO. 85-5) Buffer
Fast Blue RR Salt (Sigma Cat. NO. FBS 25) capsules

1.  Measure 50ml of dh20 into a beaker.  Add 2ml of the alk. phos. buffer
solution and one capsule of the Fast Blue RR Salt.
2.  Mix the solution well on a magnetic stirrer.
3.  Pour the solution on to slides or culture plates, without filtering.
4.  Place the coplin jar or cells in the refrigerator (4C) with a lid on ti,
and allow to stain for 10 min. to 12 hours for GMA sections, you will have
to play around with the time for culture cell plates.
5.  Remove the jar from the fridge and properly dispose of the solution, and
rinse the slides or cells very well with dh20
6.  Air dry slides and coverslip with permount.
Osteoclasts and certain other marrow cells stain purple/blue
Bone is unstained

It would probably be cleaner and easier to work with if you put your culture
cells in suspension and made cytospins or grew them on chamber slides or

-----Original Message-----
From: Ford, Judi {Path~Palo Alto} [mailto:JUDI.FORD@ROCHE.COM]
Sent: Tuesday, March 21, 2000 11:25 AM
To: ''
Subject: RE: alk phos stain in culture plates

Hi Patsy:  Thanks for replying.  I'm interested in enzyme histochemistry.
The researcher wants to do the reaction right in the petri dish of bone
marrow cultures.

> -----Original Message-----
> From: []
> Sent:	Tuesday, March 21, 2000 9:13 AM
> Subject:	RE: alk phos stain in culture plates
> are you talking about IHC or enzyme histochemistry?
> patsy
> -----Original Message-----
> From: Ford, Judi {Path~Palo Alto} [mailto:JUDI.FORD@ROCHE.COM]
> Sent: Monday, March 20, 2000 12:08 PM
> To: Histonet (E-mail)
> Subject: alk phos stain in culture plates
> Hi everyone!
> I have a question that I'm hoping someone can help me with.  One of our
> researchers wants to know of an alkaline phosphastase stain that can be
> done
> in rat bone marrow cultures. Any ideas?
> Thanks in advance for your help.
> Judi Ford

<< Previous Message | Next Message >>