Anti-CCR5 antibody (ab1673)

Dear Abcam Support,
Our order number: MDS 614616

The Anti-CCR5 antibody arrived 22-03-2012. As recommended, the tube containing the antibody was centrifuged and then aliquoted into 5 ul aliquots.
We were expecting 100 ul, and therefore approximately twenty, 5 ul aliquots. On aliquoting, we obtained 15 aliquots - suggesting a total shipping volume of around 75-80ul. I have also checked the balance & the pipette - these are working within well within their limits. Up to the point of aliquoting, the antibody was stored at 4C.
I will be grateful if you could look into this matter. I will also be grateful if you could sent us the shortfall.
PS: We have also purchased an anti-CCR2 antibody, the number of aliquots obtained were in line with the protein concentration & amount of antibody, our order number for this was MDS 614615. The same pipette was used for aliquoting both the CCR2 & CCR5 antibodies.
CC:
Kindest regards,

ANSWER:

Thank you for contacting us.
I am sorry that the vial you received did not contain the full amount of the product. We are very careful to measure out exactly the stated quantity, however, sometimes during delivery the cap can become loosened and some loss of product can occur. This is very rare but I sincerely apologize for the inconvenience this has caused you. I have issued a free of charge replacement vial with the order number of 1058239 (PO number FOCR MDS614616).
Please do not hesitate to contact us if you need anything further.

Predicted MW is 40 and papers have demonstrated that it could be as much as 40-45. Is abcam antibodty recognising correct sized band

ANSWER:

Thank you for your enquiry.

I can confirm that ab1673 recognizes a band of approximately 40kDa. and that CCR5 has a predicted molecular weight of 40524Da.

If you are continually obtaining a band of 37KDa may I suggest that you fill in our technical questionnaire. This will enable our technical team to better deal with your technical query and facilitate a quick resolution to your problem. I have added the technical questionnaire link for this antibody below.

http://www.abcam.com/index.html?section=western&pageconfig=technical&intAbID=1673&mode=questionaire

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

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM No staining

SAMPLE Paraffin sections of human spleen, human tonsil, and monkey spleen

PRIMARY ANTIBODY 1:300 incubated Overnight at 4C and 1 hour RT

ANTIBODY STORAGE CONDITIONS Recommended storage

FIXATION OF SAMPLE 10% formalin

ANTIGEN RETRIEVAL HIER: Citrate-pH6.0, EDTA-pH8.0, BORG(biocare medical EDTA-pH9.5), NUCLEAR(biocare medical Tris-pH9.5)decloaking chamber 2 min. EIER-proteinase K, 10 min @ RT.

PERMEABILIZATION STEP I use TBS/Tween prewash step

BLOCKING CONDITIONS Sniper-Casein derrived background blocker, 10 min. 3% hydrogen peroxide in PBS, 10-15min, I quench endogenous peroxidase after primary antibody incubation.

SECONDARY ANTIBODY Jackson Labs donkey anti goat IgG-HRP conjugated. 1:125 dilution, incubated 30-60 mins @ RT

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 4 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

Could you please tell me what the frequency of positive CCR5 stained cells is in human spleen paraffin sections. I have tried multiple HIER's, EIER's, different incubation times, etc... and only see a few positive cells. Is the image you supply with the primary antibody "normal tissue?" Thanks

ANSWER:

The image supplied shows a paraffin section of normal human spleen tissue.

I suspect that the blocking step with the Sniper reagent may be a problem.

Here is the protocol that we recommend.

Immunohistochemistry on Paraffin sections:

Paraffin sections of tissues fixed in 10% formalin are used.

Preparation of slides:

Place slides in 60 C oven overnight after cutting. Then follow the procedure below:

1) xylene 2x10min

2) 100% alcohol 2x10min

3) 95% alcohol 5 min

4) 80% alcohol 5 min

5) rinse in distilled water 2x2 min

6) antigen retrieval if necessary

7) Immunohistochemistry

Antigen Retrieval: Sections were treated with antigen retrieval solution and steamed for 20 minutes followed by cooling for 20 minutes.

Immunohistochemistry: Block the endogenous peroxide by incubating the sections with 3% H2O2 for 15 minutes at room temperature (RT). Wash the sections with PBS and incubate with 2% normal rabbit serum at room temperature to block the non-specific binding sites. Wash the section with PBS and incubate with primary antibody (anti-chemokine receptor) at recommended dilution for 60 minutes at RT. Wash the slides and incubate at RT with biotinylated rabbit anti-goat IgG H+L at 1-200 dilution for 30 minutes. Wash the slides and incubate with HRP-Streptavidin 1:400 dilution for 30 minutes. Wash the slides and incubate with DAB for 5-7 minutes at RT. Wash slides with PBS and counter stain with hematoxylin.

I hope that this information is helpful. Please contact me if you have any futher questions or concerns.

Thank you again. We are going to try to use ABC method and Tris Buffer next time. We also have Vectastain Universal Quick Kit. They say 0.3% H2O2 in methanol for 30 minutes. Should we use your recommended 1.6% H2O2 in TBS? And which is better to use ABC methods or Universal Immno-enzyme Polymer (UIP) methods? Please let us know.

ANSWER:

Thank you for your email. I'm not familiar with the universal immunoenzyme polymer method and so I cannot comment as to how it compares with the ABC method. As you are using the Vectastain Universal Quick Kit, I do suggest following their suggestions. Please contact us again if you have any additional questions.

Thank you for your reply.We looked your home page again after we got your reply,but we didn't understand much abut it.So we decided to answer your form of questions. We would like to you look them and return your opinion.

>. Can you please specify which CCR5 antibody you >are using? Please indicate the Abcam code number (ab....). ...ab1673

>Also, I have some general questions, the answers to which will enable me to >investigate this matter as quickly as possible:

1. Please describe the problem (high background, no staining etc). ...It has no staining about lymph cells but several granulocytes were stained.

2. On what material are you testing the antibody in IHC? •Species?... human •Cell line? •Tissue? ....Paraffin embeded sections.

3. How did you fix the samples? •Ethanol, methanol •Acetone •Paraformaldehyde •Other ...We used formaldehyde.

4. Did you apply antigen retrieval step? •Enzymatic method •Heat mediated technique...... we used EDTA buffer(pH8.0) 40 minutes (95? microwave). •Other

5. How did you block the unspecific binding sites? ...We used 3%H2O2 methyl alcohol.

6. Primary antibody •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? ...We diluted it by PBS buffer. •Incubation time, wash steps (multiple short washes are more effective than fewer longer wash steps)?...We incubeted it over night, after that we rinsed three times by PBS buffer.

7. Secondary antibody •What secondary antibody are you using? ...We used Histofine Simple Stain MAX-PO(MULTI). •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody?...not deluted •Incubation, wash steps?...30 minutes •Do you know whether the problems you are experiencing come from the secondary?...No, we don't. •What detection method are you using?

8. Background staining •Please provide an image of your staining

9. Which detection system did you use?

10. Did you apply positive and negative controls along with the samples?Please specify. ...lymph node and palate tonsil. All cells of both section were no stained.

11. Optimization attempts •How many times have you tried the IHC?...twice •Do you obtain the same results every time?...The first time, all cells were negative. The secound time, several granulocytes were stained. •What steps have you altered?...The first time, we used heat-induced epitope retrieval in citrate buffer for 25minutes. The secound time, we used EDTA buffer(pH8.0).

We used this method

deparaffinized,rehydrated, ? ?mM EDTA (pH 8.0) with high temperature( 95? microwave). ? rins PBS buffer ? 3% H2O2 methanol for 15 minutes ? rins 3 times with PBS buffer 5 minutes ? Primary antibody( ab1673) ( diluted it by PBS buffer(×300) ) over-night at room temperature ? rins 3 times with PBS buffer 5 minutes ? secondary antibody dirrect in Histofine Simple Stain MAX-PO(MULTI) kit 30minutes at room temperature ? rins 3 times with PBS buffer 5 minutes ? DAB (DAKO) within 10 minutes ? rins distilled water ? hematoxylin stain (for nucleus)

ANSWER:

Thank you for the details that you have provided. I have a few more questions for you that will help me to troubleshoot this problem:

1. I was unable to open the attachments that you sent along with your email. You mentioned in your email that "All cells of both section were no stained." Does that mean that you are not seeing any staining at all with this antibody?

2. What dilutions of ab1673 have you tried? It is recommended to use ab1673 at a dilution of 1:300 in IHC-P.

3. Have you done a positive control? Ab1673 was tested and characterized for IHC using paraffin sections of human spleen, which is recommended as a positive control.

4. What was the batch number that you received (it is located on the vial)? Also, did you order directly from Abcam or through one of our distributors?

Thank you again, and I look forward to hearing from you.