Anti-CD45 antibody (ab10558)

Our lab had recently purchased a CD45 antibody (ab10558). Our first lot we received was #******. We had very poor results with this one. We were then sent a second lot #******. We still had no positive results using this lot on either paraffin or frozen sections using both colormetric and immunofluroscence. We would like to get a refund for this antibody.

A second problem we have encountered is with a Gilz antibody (ab28945). We originally had lot #*********, which we had good results with. We then received a second lot, #*****, and have had no luck with the new lot. Is there a possibility that we can receive a different lot of Gilz?

ANSWER:

Thank you for contacting Abcam.

I am sorry about the problems you have been having with a few of our antibodies. For ab10558, as requested I have refunded the cost of the original purchase. Your credit note number is*********.

As for ab28945, we no longer have that antibody in our catalogue, so I cannot send you another lot of that one, but we do have a few other Gilz antibodies that may be suitable. If you can provide me with the order information for ab28945 and also the catalogue number of the antibody you wish to receive, I will be happy to send that out.

Also for our records, would you be able to provide a brief protocol of how you were using ab28945, including the tissue type and species.

Looking forward to your reply and helping you resolve this issue.

Our order number for the original lot we had at number: XXXX

ANSWER:

Thank you for your reply.


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.

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.

Basically, I need an antibody that works. So whatever (new lot, different CD45 antibody) you can give me that works would be great!

ANSWER:

Thank you for your reply.


This is a very popular antibody and we sell many units each month. We have several new lots available and I am happy to send you one. Can you please confirm your previous order number for this product? I will use this to arrange for the replacement to be sent asap.


I look forward to your reply so that I may process your request. Please do not hesitate to contact me if you have any additional questions.

1) Abcam product code ab10558

2) Abcam order reference number or product batch number #GR55123-1

3) Description of the problem: staining everything

4) Sample preparation: 4% PFA fixed paraffin embedded sections; 4% PFA (on ice-briefly) embedded in 15% sucrose, 7.5% gelatin for frozen sectioning

Species -Mouse

Type of sample: Fresh frozen sections, perfusion fixed frozen sections, PFA/formalin fixed paraffin embedded sections, cells in culture, other:

Sample preparation

Positive control-Spleen, uterine implantation sites, para-aortic lymph-node, estrogen treated uterus

Negative control-Non-pregnant uterine tissue

5) Fixation step

Yes/No

If yes: Fixative agent and concentration-4% PFA

Fixation time-6 hours room temp for paraffin; 20 min on ice for frozen sections

Fixation temperature

6) Antigen retrieval method- 20 minutes boiling 10mM sodium citrate for paraffin embedded sections

7) Permeabilization method: 0.1% Trition-X

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

Permeabilizing agent and concentration:

8) Blocking agent (eg BSA, serum…):

Concentration: Paraffin: Normal goat serum-10%, 0.1% Trition-X, 0.1% BSA; also tried:Normal goat serum-10%, 0.1% Trition-X, 5% BSA; Frozen:Normal donkey serum-10%, 0.1% Trition-X, 0.1% BSA

Blocking time-1 hour

Blocking temperature-RT

9) Endogenous peroxidases blocked?-Yes. 3%H2O2/Methanol for paraffin; 3%H2O2/PBS for frozen

Endogenous biotins blocked?

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

Concentration or dilution: Paraffin: 1:2000,1000,500,100,50 for paraffin; Frozen: 1:2000,1000,500,150

Diluent buffer-Diluted in blocking solution

Incubation time-O/N at 4C

11) Secondary antibody: Paraffin-sc-2040-biotin conjugated; Frozen-AlexaFluor546

Species: Paraffin-Goat; Frozen-Donkey

Reacts against: Paraffin-Rabbit; Frozen-Rabbit

Concentration or dilution: Paraffin: 1:1500; Frozen-1:500, 1000

Diluent buffer: Paraffin and Frozen-Blocking solutions

Incubation time: Paraffin and Frozen-45 min-RT

Fluorochrome or enzyme conjugate

12) Washing after primary and secondary antibodies:

Buffer-1X PBS

Number of washes-3x 15 minutes

13) Detection method-Paraffin-DAB

14) How many times have you run this staining?-5

Do you obtain the same results every time? -Yes

What steps have you altered to try and optimize the use of this antibody?-Changing AB concentration, blocking solution, and trying both paraffin and frozen sectioning

Document attachment: Attaching images of your IHC is strongly recommended and can greatly speed up our investigation of your problem.

No images to attach. Staining is in everything though. On paraffin sectioning, the uterine Natural Killer cells (CD45+) are expressing it, but so is every single other cell in the section, (not all are CD45+). In frozen sectioning, no positive staining was detected, everything was staining peri-nuclear.

ANSWER:

Thank you for your reply and for confirming the details of your protocol.

The protocol looks fine, so I'm not sure why you're not seeing the correct membrane staining with this antibody. I would like to offer you a free of charge replacement from a different lot of ab10558, a different anti-CD45 antibody, or a refund/credit note.

Please let me know which you would prefer and I would be happy to process that for you.

I sent a form for the enquiry last time when I tested the first vial. And this time happened the same. Do you want me to forward this summary?

Looking forward to your reply.

Thanks,

ANSWER:

I guess the problem you are observing is similar to the previous lot. I am sending you a different lot of this ab with order number 1048192. I hope this new lot gonna work fine if in case this lot does not work then I would suggest changing the positive control and cell type. It may be that the cells you are using as negative control do express the CD45 protein; a quick literature search would also help.

best wishes,