Methods for staining macrophages and lymphocytes with nonse specific esterase.

: This may be a very primary question, but check a few books without any information, the literature is an understatement, as if it were too primitive. However, I really do not know how to operate this method - esterase concentration, formulation and dyeing time and so on, look forward to the echo of your comrades. Thank you.
: I've done it before, I've done lymphocytes. But macrophage methods are the same and are more likely to produce results.
non-specific esterase
a fixed liquid formaldehyde-acetone buffer (PH=6.6)
Na2HPO4 20mg, 12, 50.39mg
KH2 PO4 100mg
dH2O30ml
acetone 45ml
40% formaldehyde 25ml
fixed blood cells with
ii incubator 4%
1. Pay masqueen 4g
2N hydrochloric acid 100ml added to 2n hydrochloric acid, water bath dissolved
filtration
, placed 4 degrees C, save spare
2. 4% sodium nitrite
sodium nitrite 400mg
distilled water 10ml
3. Six-a-side nitrogen pay masmoth solution
before use to take 4% sodium nitrite solution 3 ml, while shaking into 3 ml pay masmoth, and then full shock, 1min spare
4. 2% α acetate solution: take 2g alpha acetate dissolved in 100ml ethylene glycol methyl ether, placed in a refrigerator at 4 oC, avoiding light to save spare
5. M/15PH7.6 Phosphoric acid buffer
meth: KH2PO49.08g dissolved in 1000ml distilled water, b liquid Na2HPO49.47g dissolved in 1000ml distilled water 12H 2O, 23.882
13ml, B 87ml, mixed
KH2PO40.59g, Na2HPO412H2O 10.39g-500 ml
incubation liquid pre-preparation: take m/15, PH-7.6 buffer 89ml, drop by drop added hexa-nitrogen to pay masmentation 6ml, fully mixed, then dripped 2% acetate 2.5ml, fully mixed Adjust pH to 5.8-6.4
below paraffin
slice
, frozen slice can be fixed with 4% polyformaldehyde, first fixed and sliced after fixed can be
dysite wax slice
1.Will be
tissue
4 degrees C 10% neutral formaldehyde calcium solution fixed, overnight
2.Remove at least 24hr of the Arabic gum liquid from 4 degrees Cholt
3.Placed at 4 degrees C 50% acetone 1hr
4.Transfer to 4 degrees C 100% acetone 24hr (change twice), the last time left at room temperature about 1-2hr
5.Transparent 30min (two changes) in xylene
6.56 degrees C soaked wax 30-45min
7.Slices are placed at room temperature or in a 37C temperature box
dry
1-2hr.
8.Xylene dewax, after 100% acetone, 50% acetone each 2-3min, and then into distilled water
9.The post-incubation procedure is the same as the blood smear, but after 50% acetone and pure acetone each 30s-1min, xylene transparent, and then sealed
holt's sucrose liquid
sucrose 30g
aeg gum 1g
100ml
with this enzyme spread less.
1% of methyl green dyes are
used for re-
.