Question (37568) | Anti-Vimentin antibody [VI-10] (ab20346)

Go to datasheet (ab20346)

Question

Could you provide protocol or any additional informaiton specific to GMA embedding of tissue sections.

Answer

The protocol we have for GMA staining is as follows;

Place biopsy immediately in ice cold acetone containing protease inhibitors
Fix overnight -20°C
Replace fixative with acetone (room temperature) 15 min
Place biopsy in Methyl benzoate for 15 minutes (This helps infiltration of GMA into the tissue)
Prepare GMA solution A; 5% methyl benzoate in Glycol methacrylate at 4C.
Prepare embedding solution
GMA solution A (10ml) + benzoyl peroxide 70mg
Dossolve the benzoyl peroxde in solution A by gentle shaking
Embed speciomens in freshly prepared embedding solution in Taab flat bottomed capsules, placing biopsy in the bottom of the capsule and filling to the brim with resin and closing lid to exclude air.
Polymerise at 4C for 48 hours.
Store in airtight boxes at -20C.



Section cutting

Remove block from embedding capsules
Trim away the excess resin
Cut 2um sections and float out onto ammonia water (1ml ammonia in 500ml distilled water) for 1-1.5 mins.
Pick sections up onto labelled poly-L-lysine coated slides.
Dry for at ;east 1 hour at RT
Stainin imidiately



Staining

Inhibit endogenous perxodase using 0.3% H2O2 for 30 mins.
Wash slides with TBS 3x5 min
Apply primary antibody overnight
Wash slides with TBS 3x5 mins
Apply biotinylated secondary antibody or conjugated antibody as required
Wash with TBS 3x5 mins
Use DAP or AEC substares as required
Counterstain the slide with Hematixylin and mount in DPX.
Store in airtight boxes at -20C.



The following anti Fibroblast antibodies we have in catalogue

ab44971, ab11333, ab8069, ab20346, ab7752, ab92547 etc.

These antibodies unfortunatley aren't tested with GMA staining.

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