Anti-beta Galactosidase antibody (ab9361)

Thanks - my sections are8 micrometers thick. I think that I will try for your secondary antibody: ab96947.

This is just in case there is an issue with my secondary.

Let me know if you have any other recommendations,

ANSWER:

The secondary should help, if you have not been using an anti-IgY secondary.

For antigen retrieval on frozen sections, the following reference has a protocol but it utlizes a different fixation than what your rabbit tissues went through. Still, the autoclaving may work.

Application of heat-induced antigen retrieval to aldehyde-fixed fresh frozen sections.
Yamashita S, Okada Y.

J Histochem Cytochem. 2005 Nov;53(11):1421-32. Epub 2005 Jul 26

PMID:16046672

Thanks again for your help. I received the second antibody and I found that this did not work, either. I will continue to troubleshoot portions of the protocol other than your antibody. Do you have any advice regarding antibody retrieval protocols and/or preferred secondary antibodies?

ANSWER:

Thank you for contacting us. I am sorry to read that the replacement failed. Regarding protocol recommendations, an antigen retrieval step may help, though as I mentioned when we spoke, other customers have been able get staining without it. I suspect there may be lot variabilty, at least with regards to the necessity of antigen retrieval.

Can you please remind me what your samples are? I see in our records that you have PFA-perfused rabbit heart, frozen, then sectioned. Are these thin sections? A heat-mediated retrieval can be applied to sections of frozen tissue, though it may damage morphology. Have you tried this?

What secondary antibody are you currently using? You will need one specific for chicken IgY.

I look forward to your reply.

Hello,
Thank you for your quick answer.
It will be great if you can send us a new vial. The previous order information was

ANSWER:

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement for one vial of ab9361.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

Inquiry: Hello, I just ordered this chicken anti beta-galactosidase (ab9361 lot #GR76812-1) in order to detect LacZ expression in transgenic mouse brains (4%paraformaldehyde fixed, frozen, free-floating sections). I tried different concentrations (1:250 1:500 1:1000) both using a DAB reaction and by immunofluorescence (BSA1% lysine 0.1% glycine 0.1% and 5% NGS), and with or without antigen retrieval (citrate buffer 95C). I used a goat anti-chicken Dylight 488 (1:500 1hr in blocking solution). I've never been satisfied by the staining. It's really weak, with a really high background. And with antigen retrrieval and I did not see any staining at all. Could you please provide me some advices to use this antibody properly or advices for another antibody that must work in my conditions? Thank you, Amelie

ANSWER:

Thank you for your email.

I am sorry to hear this lot of ab9361 is not working as expected. I believe you have done a good job of troubleshooting and it does seem this vial is not working as expected. If you have purchased this antibody within the last 6 months I would be happy to offera replacment. We do have a different lot of ab9361 available and I'd be happy to send you a vial.

In your reply, please let me know the Abcam order reference number or PO number associated with this purchaseso I can process your request.

I've attached a picture of the vial from abcam. It's a bit small but it should help. Also I had the lot number off by one number... it's actually *****which hopefully is visible in the image.

I guess the main question that still has me stumped is that in the product description page on abcam's website (picture below) the cross reactivity of the antibody indicates Mouse, Rat, and Fruit Fly. Does this then indicate that there is a native b-gal in these organisms that is detected by ab9361?

In the past we have tried ab12081 for bgal but found it quite noisy with what appeared to be lots of punctate non specific binding. A colleagues lab recommended ab9361 as an excellent anitbody and the reviews on the website are top notch but upon closer reader it appears that some reviewers are using ab9361 on non transgenic animals including one review on fruit flies.

Quickly our protocol is as follows:
tissues removed from mouse
rinsed in PBS
soaked O/N 4C in 30% sucrose in water
rinsed PBS
embed in OCT, freeze and store frozen at -80
cut sections as needed, 6uM. Store sections at -80 till needed
Fix 10' 4C acetone
wash 3x PBS
block one hour RT in 10% serum of host of secondary + 1% fish gelatin in PBS
1ry diluted in block buffer O/N at 4C. For ab9361 1:500 dilution has given best results.
Wash next day in high salt PBS 2x5' RT then 2x5' in regular PBS
2ry for 1 hour at RT in dark. Usually Alexa Fluor 594 from molecular Probes at 1:1000 or 1:2000 dilution in block buffer. Donkey anti Chicken
wash as above
dry briefly
mount with Prolong Anitfade Gold with DAPI from Molecular Probes.
Coverslip sealed with nail polish after 24hr o/n at -20C.

ANSWER:

Thanks for the reply.

I am sorry that things are not going so well at the moment, hopefully the new vial of ab9361 that I have just sent will help. The new order number for this is *******. You should receive the new vial, early next week.

As for the expression of the B-Gal gene, my understanding for mice (and it may be different for fruit flies) is that this gene is not endogenously expressed in mice. Therefore you should only see staining in your transgenic lines. I am forwarding this enquiry onto our datasheets team for them to investigate why the species that are listed on the datasheet are present. I will get back to you as soon as I hear anything.

Please let me know if there is anything else I can help you and I will get back to you as soon as I find anything else out.