Anti-Vimentin antibody [V9] (ab8069)

Could you provide protocol or any additional informaiton specific to GMA embedding of tissue sections.
- Fibroblast marker
- Human conjuctival tissue

ANSWER:

Thank you for contacting us.

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 however if you are interested in any of these you may beeligible for our Abreview discount programme. Please click below given link;

http://www.abcam.com/index.html?pageconfig=resource&rid=11998&viapagetrap=collaborationdiscount

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Ist Vimentin oder DDR2 der bessere Marker?

ANSWER:

Anbei wie versprochen die Publikationen aus der Gruppe von Gabbiani bzw. Bochaton-Piallat (PMIDs 21868702 bzw. 17347479). Ich hoffe, diese helfen Ihnen zumindest als Ausgangspunkt für Ihre weitere Suche nach geeigneten Markern. Vielleicht ist einer von beiden ja auch zu einer Kooperation bereit? (Meine Kollegin hat beide in sehr netter Erinnerung).

Ihren Abreview haben wir gerade erhalten und ich habe ihn veröffentlicht. Damit ist der Gutscheincode freigeschalten und Sie können sich einen kostenlosen primären Antikörper Ihrer Wahl bei uns bestellen.

Falls Sie sich dafür entscheiden, den DDR2-Antikörper ab63337 zu testen, sende ich Ihnen wie besprochen den neuen Rabattcode. Das humane DDR2 hat laut BLAST eine 97%ige Sequenzähnlichkeit mit dem DDR2 des Schweins (SwissProt ID F1S212, http://www.uniprot.org/uniprot/F1S212).

DISCOUNT CODE: 100%ABR-T7T0Q Ablaufdatum: 24.04.2012

Wie am Telefon besprochen, können Sie den Gutscheincode zum Kauf eines weiteren Antikörpers einsetzen, sobald Sie uns ein Abreview für diesen Antikörper eingereicht haben. Der Discountcode hat die Nummer 100%ABR-T7T0Q und kann zum Kauf weiterer Produkte eingesetzt werden.

Der Code wird gültig, sobald Sie uns ein Abreview für diesen Antikörper über den Test im Schwein eingereicht haben. Bitte geben Sie diesen Code auch im Abschnitt “Additional Notes” des Abreviews mit an, so dass wir wissen, dass sich dieses Abreview auf die Gutscheinaktion bezieht. Der Code wird dadurch aktiviert. Bitte kontaktieren Sie bei der nächsten Bestellung unsere Kundendienst mit dem Code und der ursprünglichen Bestellnummer.

Bitte zögern Sie nicht, sich wieder an uns zu wenden, falls Sie weitere Fragen haben. Wir freuen uns auf Ihr Abreview, egal ob Ihre Ergebnisse positiv oder negativ sind. Die Bedingungen dieses Angebotes befinden sich unter dem folgenden Link: www.abcam.com/collaborationdiscount

Ich wünsche Ihnen ein frohes Fest und alles Gute im Neuen Jahr sowie viel Glück und Erfolg für Ihre Forschung!

Thank you for your response. Please see below for the reply from our customer:

The spec sheet states that the ab contains only azide and BSA Could you please find out what else is in the TC medium, especially animal serum. Hope to here form you soon.

ANSWER:

Thank you for your enquiry.

The culture medium used was RPMI 1640 with 10% Foetal Bovine Serum.

If you have any additional questions or remarks, please do not hesitate to contact me.

We have a customer here, who purchased the above antibody on the 3rd of November, '09 and he received it last week.

The purchase order number is PO-06176 and the lot number is lot# 7049632. He says that the appearance of the antibody is a colour red/pink and he wants to know what is making it this colour. Could you please advice the reason for the antibody's appearance and whether it is normal? Hope to hear from you soon.

ANSWER:

Thank you for your enquiry.

The antibody was harvested from tissue culture supernatant and placed in PBS buffer, therefore some of the culture medium (like DMEM, etc) may be included in the product. Please rest assured that the red/pink color is normal and that the culture medium does not affect the product's quality.

I hope this information will help resolve your customer's concerns.

If there is anything else that I can help you with, please do not hesitate to contact me.

I am seeking an antibody specific to human cells, that does not cross-react with mouse (for some xenograft confirmation). I was told by a colleague (somewhat vaguely) that mitochondrial antibody could specifically identify all human cells, without detecting mouse. Do you have an antibody in your catalogue that would fit the bill which you could recommend for my application (not having any experience of mitrochondrial proteins I'd not like to hazard a guess at one in particular being ubiquitously expressed)?

ANSWER:

I know of one antibody that may be useful for your study. It is an anti-vimentin antibody, clone V9, catalogue ID ab8069:

http://www.abcam.com/index.html?datasheet=8069

Species-specific reactivity of this clone is described in Bohn et al, 1992, PMID: 1612114. I have no experience with the other clone the paper mentions, 3B4.

The issue you may have with this antibody is background staining caused by a secondary anti-mouse IgG binding to endogenous mouse IgG. My lab worked around this by biotinylating the antibody, then using it with and avidin-biotin-enzyme (HRP) complex, also know as ABC reagent.

I am not sure what mitochondrial protein your colleague was referring to, but if you find out the name, our search engine found in the upper left-hand corner of our homepage will allow you to filter by species reactivity and application.

I hope this helps. Please contact us if you have any other questions.