Anti-S100 antibody [8B10] - Astrocyte Marker (ab8330)

Inquiry: I wist to have an astrocyte marker for IHC-wholemounts. please suggest me suitable antibody.

ANSWER:

Thank you for your inquiry.

I am happy to confirm that we do have an antibody in our catalogue that can be used as astrocyte marker and is tested and guaranteed for IHC- whole mounts:

ab8330

http://www.abcam.com/index.html?datasheet=8330 (or use the following: http://www.abcam.com/index.html?datasheet=8330).

Please follow the above link to review the datasheet.

Please do not hesitate to contact me again with any further questions.

1) Abcam product code: ab8330, ab7388, and ab955
2) Abcam order reference number: XXXXXX
3) Problem: lack of signal compared to no-primary control
4) Sample prep: mouse brain, heart, lung, IHC-P for positive control; negative control is same tissue with no primary antibody
5) Fixation step: yes, 10% formaldehyde fix, 10-20 minutes room temperature
6) Antigen retrieval method: sodium citrate buffer (1.92g citric acid, 1N NaOH, 1000mL water, 0.5mL Tween-20 pH 6.0) at 95*C for 30 minutes, allow to cool for 45 minutes
7) Permeabilization: There is .05% Tween-20 in the antigen retrieval step and 0.1% Triton X-100 in the block step
8) Block: 1% BSA + 0.1% Triton X-100 in HBSS, 30 minutes, room temp
9) Endogenous peroxidases and biotins blocked? No
10) Primary antibodies: ab8330 at 1/1000, ab7388 at 1/250, and ab955 at 1/200, all in the block buffer listed above, for 1 hour room temp.
11) Secondary antibodies: Molecular Probes goat anti-mouse IgG alexa fluor 568, Molecular Probes A11077 goat anti-rat IgG alexa fluor 568, 1/1000 in block buffer listed above, for 1 hour
12) Washes: yes, there are 3x 5-minute washes after the primary and secondary antibody steps
13) Detection method: fluorescence using a Nikon confocal microscope
14) I have tried this particular staining once using established protocols in the lab. Since Ab7817 worked fine, I am confident with the protocol.

Thanks!

ANSWER:

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results. The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab955 since this antibody has been tested in mouse paraffin-embedded tissue sections.


For the fixation, we generally recommend between 18 - 24 hrs. It may be just that your sections are underfixed.


For antigen retrieval, was this performed in the microwave? We generally recommend 20 mins, but it's best to optimize between 15 - 30 mins. When the 20 mins has elapsed, you can run cold tap water into the vessel for 10 mins.


If you want to permeablize the sections, you should do a 0.1% Triton-X 100 in PBS treatment as a separate step and omit the Triton X-100 from the blocking buffer.


For blocking, have you tried5-10% normal goat serum instead of BSA? You should also put this in PBS.


The most important thing to try wouldbe to test a1/100 dilution of ab955 overnight at 4C instead of 1/200 for 1 hr RT. You should do this incubation in 1% BSA in PBS.


For the s100 antibody ab8330, would you be able to run a positive control with rat tissues since this antibody isn't tested in mouse?


For the CD31 antibody, ab7388, we know that this does not work well with PFA fixation (see Abreview linkedbelow). We have heard that it works wellwhen used on frozen sections of mouse skin that werefixed in methanol:acetone for 10 mins. Would you be able to try some frozen methanol or acetone fixed sections as a positive control since IHC-P does not work.


http://www.abcam.com/index.html?datasheet=7388&tab=abreviews&intabreviewid=23226

Should the suggestions not improve the results, please do let me know.

In the event that a ab955 is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund for this tested antibody.

I hope this information is helpful, and I thank you for your cooperation.

I had an older batch of this antibody, which worked fine, but the new batch does not. Could you please help me? Could you also please let me know the epitope of the antibody?

ANSWER:

I have contacted the lab regarding the epitope for ab8330 and received the following answer.

"Unfortunately we have not determined the epitope for anti-S100 ab8330. The only information we have is that according to our internal tests ab8330 recognizes alpha-beta and beta-beta forms. It does not recognize the alpha-alpha form or alpha subunit only."

I am sorry, that I could not give you more detailed information and look forward to receiving your questionnaire.

I am interested in developing a bead-based sandwich ELISA for S100 using the antibody pair ab8330(8B10) and ab8332(4D2). I have encountered difficulties with other antibodies/pairs for this target, specifically a weak signal or no signal at all. Do you have any protocols available that are specific to this antibody pair as conventional ELISA protocols have not worked thus far. Thank you for your help.

ANSWER:

Thank you for your enquiry and also your patience.

I would actually recommend that you pair ab8330 and ab10203 or ab8334 and ab10203. I will update the datasheets to reflect this also. I have also included our protocol for use with these antibodies. Either pair of antibodies should work for you and I would be interested in hearing your feedback if you purchase these antibodies. Here is our protocol:

Two-step “sandwich” immunofluorometric assay for S-100 protein:

Assay should be performed in 96-well microtitration plates. Capture Mabs are unlabeled, detection Mabs are labeled with the stable chelate of Eu3+.

1st step Capture Mabs (1 µg/well; 100 µl/well) are incubated in PBS (10 mM KH2PO4, 150 mM NACL, pH 7.4) in 96-well plate for 60 minutes at room temperature with gentle shaking.

2nd step After washing (DELFIA® washing solution, two times) the mixture of antigen (30 µl/well, standards, dissolved in normal human serum or testing samples) and detection Mabs (100 µl/well, in DELFIA® Assay Buffer) are added. After 30 minutes incubation at room temperature the plates are washed 6 times, LANFIA enhancement solution (0.2 ml/well) is added, and incubation is continued for 3 minutes at room temperature with gentle shaking. The fluorescence (cps) is measured with the 1234 DELFIA® Plate Fluorometer.

The method described has a detection limit below 0.05 µg/L, and the linearity range is 0.1–500 µg/L.

I hope this information helps, please do not hesitate to contact us if you need any more advice or information,

We are well aware of the many factors that may interfer with a succesfull protein separation, blotting and immunostaining. We have tested in two labs the procedure without firm results.

May we ask you: 1) to provide evidence that the antibody in question recognizes specifically the S100B dimeric protein on blots 2) to describe the underlying method for this evidence.

ANSWER:

Thank you for your reply.

We would like to confirm that ab8330 recognizes both the alpha-beta and beta-beta forms of S-100 in Western blotting. A western blotting image with the legend can be seen on our on-line product datasheet.

Western blotting protocol - protein samples are separated by 10-20 % gradient SDS gel electrophoresis and transferred to nitrocellulose membrane (Biotechnology, 1992, 24: 145-9). To block nonspecific binding of the proteins, the nitrocellulose membrane was incubated for 15 min at room temperature in phosphate buffered saline, containing 1 g/L Tween 20 and 5 g/L casein. The membrane was then incubated for 1 hour in the same buffer containing 0.02 g/L anti- S100 antibodies and 1 g/L casein. After the nitrocellulose membrane was washed with phosphate buffered saline containing 1 g/L Tween 20, it was incubated for 2 hours with horseradish peroxidase-labelled anti-mouse antibodies, and the binding of Mabs was visualized by incubation with diaminobenzidine.

We hope this information will be useful for you.