Staining of Blood and Bone Marrow slides

[Lisa]

Can anyone recommend a commercially prepared Wright stain (or Leishman
stain) used to stain metaphases from Cytogenetic blood and bone marrow
slides? We currently make our own Wright's stain from powder but would
like to purchase
premade stain. We have tried Giemsa but do not like the results
obtained for our slides. (We do use Giemsa for our in situ cultures
with no problems.)
Also, if you could share a protocol for banding and staining with the
premade stain that would be much appreciated.

Thanks,
Lisa

[oxforduni]

Hi Lisa, we use Leishman stain with good results.
The Optimum time in the stain appears to be between 2 to 4 minutes .
Note:
Leishmans stain is a paler stain when compared to giemsa. The time in
trypsin is between 5-30 seonds. The following website will help you
learn more about this technique (see below). Leishman stain will give
you clearer
staining than giemsa, I have found this especially with our marrows.
http://humgen.wustl.edu/hdk_lab_manual/cam/cam4.html
We buy our solution from Merck...they may sell wrights stain too.

[julie]

Hello Lisa, our wright stain is from Merck (cat number 1.01383.0100)
100ml bottle.
Merck also supplies 500ml and 2.5L

Our Wrights G Banding method:
1.Place two slides horizontally on a rack over a sink
2.Add 1ml Wrights stain to 3ml buffer , mix rapidly with a pipette and
evenly pipette onto two slides
3.Stain for 3-5 minutes.
4.Rinse off the stain gently under running tap water for 5 seconds
5.Dry rapidly using a hair dryer.
6.Examine under the microscope. If the staining is under (chromosomes
are slightly bloated and pale with only landmark bands visible) simply
repeat the procedure over again. If its overdone (chromosomes are dark
and appraoch solid staining) destain the slide by leaving it in each
of the destaining alcohols* . Dry the sliden and stain it again for a
shorter period.
7.Mount with DPX.


*Destainig alocohols procedure
(a) 70% ethanol (1 min)
(b) 70% ethanol (1 min)
(c) 95% Ethanol containing 1% HCL (1 min)
(d) Methanol (1 min) slides need not be thoroughly dried between
washes.

Note1: Wright's Stain Buffer add 0.06M disodium hydrogen
orthophosphate to 0.06M potassium dihydrogne orthophosphate to give a
pH 6.8. The approximate amounts are 99ml and 101ml, respectively.

Note2: Making wright's stain from powder (Merck cat#:1.09278.0025)
weight 2.5g wright's stain and dissolve to 1L methanol. Stire 1 hour
and then double filter through Whatman no1 filter paper into a brown
glass bottle.
Store 2-3 weeks away from sunlight before use.
Also ensure that the bottle is air tight.

On Mar 31, 11:34 pm, Lisa <lxdav...@sentara.com> wrote:

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[LISA DAVENPORT]

Thanks for the information. I appreciate the feedback and the protocol.