[Histonet] CAE and Mature Mast cells look at procedure
Okay my histoheroes and heroines,
One of the investigators here asked for a CAE designed for plastics(but supposedly works on FFPE). I did this protocol, but it isn't working. I am having a rough time because I am not used to making mistakes(kidding). She needs mature mast cells only, I gave her 7 different Mast cell procedures that all worked very well(even the dominicis), and one Alcian Blue Safranin that stains Heparin containing Mast cells red Amine containing Mast cells blue. So 2 questions are:
1-Do mature cells stain differently?
2-Does anyone have a decent CAE they can share? Here is the one I used and the control is fresh rat skin and all the other procedures worked on that block.
Chloroacetate Esterase (CAE) Staining
Preparation of Solutions
1. New Fuchsin Solution
New Fuchsin 1g
2N hydrochloric acid (HCl) 25ml
2. 4% Sodium Nitrite Solution
Sodium Nitrite 1g
3. Phosphate Buffer (0.1M, pH 7.6)
A. 0.2M solution of monobasic soduim phosphate (27.8g NaH2PO4 in 1L H2O)
B. 0.2M solution of dibasic sodium phosphate (28.4g Na2HPO4 in 1L H2O)
--> To make pH 7.6 Solution: mix 6.5mL of A, 43.5mL of B, and 50mL of ddH2O.
4. Naphthol AS-D Chloroacetate Solution (Store at -20°C)
Naphthol AS-D Chloroacetate 10mg 100mg
N-N Dimethyl Formamide 5ml 50ml
Staining Solution (make fresh before each use)
In a single vial: 1) mix New Fuchsin and 4% Soduim Nitrite
2) add phosphate buffer and mix
3) add Naphth AS-D Chloroacetate Soultion and mix well
Use 1 ml freshly mixed solution for approx 4 tissue slides
New Fuchsin 2.5µl 5µl 12.5µl 25µl
4% Sodium Nitrite 2.5µl 5µl 12.5µl 25µl
Phosphate Buffer 1ml 2ml 5ml 10ml
Naphthol AS-D Chloroacetate 50µl 100µl 250µl 500µl
1. Stain with fresh staining solution for 20 min.
2. Wash with running tap water (For plastic sections use wet cotton swab wipe off dusty residue).
3. Counterstain with Gill's II hematoxylin for 20 to 45 seconds (or methyl green for 30 seconds).
4. Wash with warm tap water to blue (longer will result darker blue, or dip into 50% of lithium carbonate for 5 - 20 seconds for deeper blue, then rinse with water).
Dry and mount with mounting medium. (For paraffin sections, quick dehydration before mounting).
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