Anti-S100A4 antibody (ab27957)

Dear Sir or Madame,

Product: ab27957

We have recently purchased the antibody anti-S100A4 (Ab27957). It doesn't work in our hands as it binds aspecifically in western blot experiments (no bands around 12 KDa). Attached is a WB image (PVDF membrane was used, Dako swine-anti-rabbit as secondary) of adenovirus-treated endothelial cells undergoing endothelial to mesenchymal transition.

It is possible to be refunded for this order?

Best regards

ANSWER:

Thank you for taking the time to contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been succesful.

I would like to reassure you that this antibody is tested and covered by our guarantee forWB and for Mouse, Rat, Horse, Cow, Cat, Dog, Human samples. In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

Before deciding how to proceed, I would like to investigate this particular case further for you, and also obtain some further information for our quality records.

In order to do this, I have enclosed a questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily.

Thank you for your time and cooperation. We look forward to receiving the completed questionaire.

Order Details

Antibody code:

Problem
Choose: Non-specific band Multiple bands No signal or weak signal High background

Lot number

Purchase order number
or preferably Abcam order number:



General Information
Antibody storage conditions (temperature/reconstitution etc)


Description of the problem (high background, wrong band size, more bands, no band etc.)


Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)


Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)


Amount of protein loaded


Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)


Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)


Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Detection method (ECL, ECLPlus etc.)


Positive and negative controls used (please specify)



Optimization attempts (problem solving)
How many times have you tried the Western?



Have you run a "No Primary" control?
Yes No

Do you obtain the same results every time?
Yes No
e.g. are the background bands always in the same place?


What steps have you altered?


Additional Notes:


Image:
We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to asess the results.

Hi there.

I recently ordered the S100A4 antibody (ab27957) and when it came in it was placed into the freezer for storage and not at 4 C. What sort of impact do you think it this going to have had on the antibody?

Kind regards

ANSWER:

Thank you for contacting us.

There will be a minimal impact on the antibody and I expect no problem.

Indeed, many antibodies are stored at -20 C, depending also on the buffer and on the conjugation. This antibody is in a normal buffer and unconjugated, therefore I do not expect any problem if the antibody has been frozen once. We indicate to store this antibody at 4 C asthis is how the laboratory has tested it.Please see also our antibody storage guide for further information. http://www.abcam.com/index.html?pageconfig=resource&rid=10795

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

I would like to test ab27957 for use on pig samples.

ANSWER:

DISCOUNT CODE: ***
Expiration date: July 7th, 2012

I am very pleased to hear you would like to accept our offer and test ab27957 in pig. This code will give you 1 free primary antibody before the expiration date. To redeem this offer, please submit an Abreview for pig and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.

Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code.

Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

The terms and conditions applicable to this offer can be found here: http://www.abcam.com/collaborationdiscount.

1) Abcam product code ab27957

2) Abcam order reference number or product batch number

3) Description of the problem

A while ago we ordered your S100A4 antibody ab27957 as we need to stain fibroblasts in tissue sections. As S100A4 is fibroblast specific this antibody should be useful for us. However I have some problems with staining fibroblasts.

When cytospins are stained with just single cells, 3T3 cells (pos control) are stained and ECs (neg control) are not stained. Till so far we are happy. But when I stain tissue sections, all smooth muscle cells are stained too. It looks like the staining is specific, it just stains the wrong cells. We used human and ovine blood vessels and ovine small intestine as positive controls. Frozen sections of 10 um are stained with concentrations between 1:100- 1:600 and in all sections the smooth muscle cells are stained instead of fibroblasts only.

4) Sample preparation:

Species human and ovine

Type of sample: Fresh frozen sections, cytospins of cells

Sample preparation

Positive control

For tissue sections: human and ovine vein (human vena saphena and ovine vena jugularis, ovine small intestine.

For cytospins: 3T3 cells

Negative control

For tissue sections: human and ovine vein (human vena saphena and ovine vena jugularis, ovine small intestine.

For cytospins: ovine endothelial cells

5) Fixation step

Yes, ice cold aceton for 10 min at 4 degrees

6) Antigen retrieval method No

7) Permeabilization method:

Did you do a permeabilization step (details please) or add permeabilizing agent in any dilution buffers?

Permeabilizing agent and concentration: 0.1% saponin in PBS as a permeabilizing buffer and as a dilution buffer for the antibody

8) Blocking agent (eg BSA, serum…): 5% BSA in PBS for 30 minutes at room temperature

9) Endogenous peroxidases blocked? No

Endogenous biotins blocked? No

10) Primary antibody (If more than one was used, describe in “additional notes”) :

Concentration or dilution 1:100 – 1:600

Diluent buffer 0.1% saponin in PBS

Incubation time overnight at 4 degrees

11) Secondary antibody:

Species: Goat IgG

Reacts against: Rabbit

Concentration or dilution 1:200

Diluent buffer 0.1% saponin in PBS

Incubation time 30 minutes at room temperature

Fluorochrome or enzyme conjugate alexa 488

12) Washing after primary and secondary antibodies:

Buffer PBS

Number of washes 3x 5 minutes under gentle agitation

13) Detection method Fluorescent microscope

14) How many times have you run this staining? About ten times

Do you obtain the same results every time? yes

What steps have you altered to try and optimize the use of this antibody? Different concentrations of first antibody. Different cells and tissue sections as controls. Secondary antibody shows no unspecific staining, thus this is already optimal.

Added pictures:

1. ovine intestine dilution 1:400. Blue is DAPI, green is where the S100A4 antibody is bound.

20x magnification: Smooth muscle cell layers of small intestine are stained (both longitudinal and circular directed smooth muscle cells). Other cells on the left of the smooth muscle cells are not stained.

10x magnification: small blood vessels around the intestine are stained.

2. Human blood vessel (H001 10x magnification). Blue is DAPI. Green is where the S100A4 antibody is bound. Smooth muscle cells in the media are stained, and also smooth muscle cells of the surrounding small vessels. Expected was that just fibroblast in the adventitia would stain for this antibody.

ANSWER:

Thank you for getting back to me and for providing some further information on the immunostaining.

As I understand from your e-mails that ab27957 works well in ICC/IF using cytospin preparations. However, on tissue sections (human and ovine blood vessels and ovine small intestine) non-specific signal could cause some difficulties.

After reading through the detailed protocol you kindly forwarded to Abcam, I would like to make the following comments/suggestions:

1) Fixative:

Acetone is a fixative but it also permeabilizes the cellular membranes. It does not require further permeabilization step. I would recommend omitting the additional permeabilization with 0.1% saponin to see if the signal is getting more specific or not. Alternatively, you could try different fixatives such as ice-cold ethanol, methanol or even PFA.

2) Incubation with the primary antibody:

It may be worth incubating the samples with ab27957 for shorter period of time i.e. 1 or 2 hrs.

3) Dilution buffer:

Try to use TBS-T (with 5% BSA) for diluting the primary and the secondary antibody instead of 0.1% saponin in PBS.

I hope this helps and if I can assist further, please do not hesitate to contact me.

Can you tell me which cell type this does product, stain in tonsil tissue?

ANSWER:

Thank you for your enquiry.

In tonsil tissue, you will see labeling of T-lymphocytes and blood plasma cells.