Anti-HSV1 + HSV2 gB antibody [T111] (ab8232)

Thank you for your wonderful assistance!
Would you be able to advise me whether Ab8232 is appropriate in this case, and what other products would you recommend?
Please refer to the question from our customer below:
“With regards to anti HSV-2 antibdy, I'm going to stain mouse tissue with anti HSV-2 antibody (Immunofluorescence, IF). The only antibody we have got in the lab is Rabbit anti herpes simplex virus antibody from Dako. In addition to this antibody I wanna stain this tissue with another Rabbit antibody which makes it very difficult or even impossible to put both antibodies in one sample. So, I'm looking around to find another alternative for anti HSV-2 antibody. I saw an antibody in Abcam website which might be relevant.
It'sAnti-HSV1 + HSV2 gB antibody [T111] (ab8232).
I'm not sure if this antibody could work on my sample nor there is any other antibody in your website.
So, Could you please look around of your products to see if there is something that might help me to get this IF done?”
Looking forward to your response!
Have a nice day J
Kind regards,

ANSWER:

Thank you for contacting us.

The following anti HSV2 antibodies have been tested in IF and are fully guaranteed.

ab21112, ab9534, ab934, ab23502, ab20971, ab49555.

It seems that you are planning for double Immunostaining Right!, then you will need one rabbit and one mouse antibodies. Mouse antibodies we have are ab23502, ab8258, ab49555, ab861.

Please check the catalogue you will be able to find the right product.

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

Use our products? Submit an Abreview. Earn rewards!
http://www.abcam.com/abreviews

Order Details Antibody code:Ab6506

Lot number: GR7558-2

Purchase order number or preferably Abcam order number:

General Information Antibody storage conditions (temperature/reconstitution etc)
-Kept at -20C

Description of the problem (high background, low signal, non-specific staining etc.)
-no staining, mainly non-specific since control without virus gave same background

Sample (Species/Tissue/Cell Type/Cell Line etc.)
-used VERO cells from Tissue Culture

Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.)
-fixed in paraformaldehyde (30 min at RT)

Antigen retrieval (Enzymatic method, Heat mediated technique etc.)

Permeabilization step
-0.5% triton 3 min

Blocking conditions (Buffer/time period, Blocking agent etc.)
-1%BSA in PBS

Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
-from 1:6000 to 1:100 in 1% BSA for 1 hour at RT
-followed by 3 washes in PBS

Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
-Alexa-488 anti-mouse 1:500, 45min at RT
-Wash 3x in PBS


Detection method
-Confocal microscopy

Positive and negative controls used (please specify)
-used DAPI, used infected vs non-infected cells

Optimization attempts (problem solving) How many times have you tried the IHC?
- Have only tried it once will repeat again and then compare resutls
-also will try a WB since the two publication showed WB images


Thank you,

Claudia Arana



Have you run a "No Primary" control? Yes

Do you obtain the same results every time? Yes




On Thu, Jan 26, 2012 at 3:37 PM, <mailto:technical@abcam.com> wrote:



Dear Claudia,

We have an answer to your inquiry:
Thank you for your enquiry.

I am sorry to hear you have had difficulty obtaining results from this antibody. I can confirm the ICC image provided was from external testing, and regrettably we do not have more information about how it was obtained on this occasion.

Regarding a publication, I hope the following article could be helpful:

Bystricka M, Zatovicova M, Petrikova M, Solarikova L, Russ G, Ziegler T: Monoclonal antibodies suitable for type-specific identification of herpes simplex viruses by a rapid culture assay. Acta Virol. 1999 Dec;43(6):399-402.

I would like to reassure you that this antibody is tested and covered by our 6 month guarantee in ICC. In the event that a product is not functioning in the applications 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.

I would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily.

I would appreciate if you could also provide an image which would help us to assess the results.

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

Order Details Antibody code:

Lot number:

Purchase order number or preferably Abcam order number:

General Information Antibody storage conditions (temperature/reconstitution etc)

Description of the problem (high background, low signal, non-specific staining etc.)

Sample (Species/Tissue/Cell Type/Cell Line etc.)

Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.)

Antigen retrieval (Enzymatic method, Heat mediated technique etc.)

Permeabilization step

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

Positive and negative controls used (please specify)

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



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

Do you obtain the same results every time? Yes No

What steps have you altered?

Additional Notes

We would appreciate if you are also able to provide and image which would help us to assess the results
Help us improve our service. Rate your experience with us today. http://www.abcam.com/index.html?pageConfig=technicalSurvey&intCCEID=3457693

Your original inquiry to Abcam:
-------------------------------------------

Hi,

We have recently ordered the gB AB10B7 and would like to know what conditions were used for the FITC-image displayed on the antibody page. The two reference papers are for WB images we will like to use this antibody for IF. The antibody dilution used in the IF image is 1:100, however I have not been able to get a signal in my vero cells. Could you please specify, length of infection, buffers used for staining. How long post infection were cells fixed, etc.

Thank you.

Claudia Arana




----------------------------

Claudia Arana

PIKE Pharma GmbH

Wagistrasse 27a

8952 Schlieren

Switzerland

tel:%2B41%2044%20200%202114





-------------------------------------------


Best regards,
Kate

Kate Hayes
Scientific Support Supervisor
Abcam plc
http://www.abcam.com


Abcam Customer Services and Technical Support Team

http://www.abcam.com/technical
[CCE3457693]

ANSWER:

Thank you for taking the time to complete our questionnaire.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.
Reviewing this case, I would like to offer some suggestions to help optimise the results from ab8232. I would also appreciate if you can confirm some further details:

1. I would appreciate if you are able to provide an image, which would help me to assess the results.

2. Could you confirm further details of the fixation in paraformaldehyde? I can recommend to try 4% PFA for 10 minutes only, this will help to ensure there is not too much crosslinking.

3. For permeabilization, 0.5% Triton is quite high and could disrupt proteins and epitopes. I can recommend to try 0.2% for 10 minutes.

4. Try a higher concentration of blocking agent, for example 5% BSA. How long was the block step? I can recommend to try 30 minutes to 1 hour.

5. I can recommend to add a gentle detergent such as 0.2% Tween to the wash buffer. This would help to wash away any excess antibody. This can also be added to the antibody dilution buffer to help keep the antibody solubilised.

6. Could you confirm if the secondary antibody is working well with other primary antibodies? I can recommend to consider including a no primary control which will help to asses if the background is from the secondary antibody. The concentration of the secondary may need to be reduced to help optimize the results.

In the event that a product is not functioning in the applications 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.

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

Hi,

We have recently ordered the gB AB10B7 and would like to know what conditions were used for the FITC-image displayed on the antibody page. The two reference papers are for WB images we will like to use this antibody for IF. The antibody dilution used in the IF image is 1:100, however I have not been able to get a signal in my vero cells. Could you please specify, length of infection, buffers used for staining. How long post infection were cells fixed, etc.

Thank you.

ANSWER:

Thank you for your enquiry.

I am sorry to hear you have had difficulty obtaining results from this antibody. I can confirm the ICC image provided was from external testing, and regrettably we do not have more information about how it was obtained on this occasion.

Regarding a publication, I hope the following article could be helpful:

Bystricka M, Zatovicova M, Petrikova M, Solarikova L, Russ G, Ziegler T: Monoclonal antibodies suitable for type-specific identification of herpes simplex viruses by a rapid culture assay. Acta Virol. 1999 Dec;43(6):399-402.

I would like to reassure you that this antibody is tested and covered by our 6 month guarantee in ICC. In the event that a product is not functioning in the applications 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.

I would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily.

I would appreciate if you could also provide an image which would help us to assess the results.

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

Order Details Antibody code:

Lot number:

Purchase order number or preferably Abcam order number:

General Information Antibody storage conditions (temperature/reconstitution etc)

Description of the problem (high background, low signal, non-specific staining etc.)

Sample (Species/Tissue/Cell Type/Cell Line etc.)

Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.)

Antigen retrieval (Enzymatic method, Heat mediated technique etc.)

Permeabilization step

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

Positive and negative controls used (please specify)

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

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

Do you obtain the same results every time? Yes No

What steps have you altered?
Additional Notes

We would appreciate if you are also able to provide and image which would help us to assess the results