Anti-CD68 antibody [FA-11] (ab53444)

I would like to answer those questions.
1. For antigen retrieval: cooking in citrate buffer for 20 min, leave in citrate buffer for 10 min and cool by keeping it outside for 5 min.
2. The blocking buffer has also been reduced to 3% BSA. But no use.
3. Regarding Rat AP-polymer kit, I don't know which information do you need. Because we follow the kit protocol.
4. We have stained our slides with CD3, CD8 and other antibodies also by following the same protocol and just this one is not working.

ANSWER:

I will issue you replacement or credit note if you wish, however, i would just like to make some comments:

I was asking about the Rat AP-polymer kit because we usually like to check the compatibility with our products as a few cause trouble sometimes and we than can simply recommend a different one.

Please keep in mind that you cannot compare two different antibodies against two different targets with each other. Every antibody needs its individual optimization.


Thank you again for your time and cooperation. Please let me know on how you would to proceed.

I have attached the answers for anti-CD68. Do, I have to give for anti F4/80 also?

Have a nice day



1) Abcam product code ab 53444

2) Abcam order reference number or product batch number GR76629-1

3) Description of the problem : No staining

4) Sample preparation:
Species Mice
Type of sample: Fresh frozen sections, perfusion fixed frozen sections, PFA/formalin fixed paraffin embedded sections, cells in culture, other: Formalin fixed
Sample preparation paraffin embedded
Positive control: spleen
Negative control

5) Fixation step
Yes/No No
If yes: Fixative agent and concentration
Fixation time
Fixation temperature

6) Antigen retrieval method: Cooking with citrate buffer pH 6 or cooking with HIER-T-EDTA pH 9

7) Permeabilization method:
Did you do a permeabilization step (details please) or add permeabilizing agent in any dilution buffers? No
Permeabilizing agent and concentration:


8) Blocking agent (eg BSA, serum…):
Concentration BSA 10%
Blocking time 1 hour
Blocking temperature room temperature

9) Endogenous peroxidases blocked?
Endogenous biotins blocked?

10) Primary antibody (If more than one was used, describe in “additional notes”) :
Concentration or dilution : 1:20, 1:50 and 1:100
Diluent buffer Antibody diluent from IVD
Incubation time: overnight

11) Secondary antibody: Rat AP-Polymer kit used
Species:
Reacts against: rat
Concentration or dilution
Diluent buffer Antibody diluent from IVD
Incubation time
Fluorochrome or enzyme conjugate

12) Washing after primary and secondary antibodies:
Buffer: TBS
Number of washes: 6 times 20 minutes

13) Detection method: Rat AP-POLYMER kit

14) How many times have you run this staining? Many times even with different lot of mice lungs
Do you obtain the same results every time? Yes
What steps have you altered to try and optimize the use of this antibody?

Document attachment: Attaching images of your IHC is strongly recommended and can greatly speed up our investigation of your problem.
No pictures because there was no staining at all.

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 fromab53444 Anti-CD68 antibody [FA-11]. I would also appreciate if you can confirm some further details:

1. How long do you perform the antigen retrieval? Do you perform a time series?

2.Reduce the blocking buffer down to 3% BSA, as the problem is not a high background, but no signal. 30 minutes blocking should be sufficient as well.

3. Could you please give me more information regarding the secondary antibody: Rat AP-Polymer kit?

4. In order to avoid a washing of the antibody, please decrease the washing after each step down to 3 time a 10 min.



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

In the event that a product 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.

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

ab2350: IHC-Fr with mouse lung and liver (20 and 90 um sections)
liver: not intense but correct staining
lung: no signal
Ab: 1/50, 1/25 o/n 4 degree
blocking: 55 serum
secondary: Alexa488, works well
perm step: TX100
fix step: acetone 10 min
send IHC-Fr protocol for ab27853

ab53444: IHC-Fr with mouse lung and spleen
high background
Ab: 1/50
suggested: use less antibodies and incubate shorter, block longer and more (10% serum)
send IHC-Fr protocol

ANSWER:

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry the product ab2350 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 with the order number xxx with ab27853.

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.

As for the IHC-Fr protocols:


For ab27853, I will need to check with the supplying lab if they have any details on that. As far as I know, it was reported to them by other researchers that the antibody works in IHC-Fr. I will let you know what I find out.

I have attached our IHC-Fr protocols to help you further in the meantime.


For ab53444,we have the following protocol on file:

Indirect Immunostaining of Frozen Tissue Sections

For use with unconjugated monoclonal and polyclonal antibodies.
Please note that a certain level of technical skill and immunological knowledge is required for the successful design and implementation of these techniques - these are guidelines only and may need to be adjusted for particular applications.
Reagents
1. 0.3% H2O2‎, 70% methanol in TBS
1 ml of 30% H2O2‎ per 100 ml methanol/TBS.
2. TBS stock solution (10x)
NaCl, 87.66 g
Tris base, 60.55 g
Distilled water, 1 liter
Adjust pH to 7.4 using concentrated HCl.
Method
In most cases, we recommend that tissues are snap-frozen in liquid nitrogen, then prepared as 4-6 μm sections using a cryostat.
Allow sections to air dry for at least 1 hour.
Fix sections in dry acetone for 15 minutes. Allow to evaporate for 10 minutes.
Block endogenous peroxidase (if necessary) by immersing slides in 0.3% H2O2‎ in 70% methanol/TBS for 30 minutes. Wash once in TBS.
Incubate sections for 10 minutes in 10% normal serum from the species in which the secondary was raised. Tap excess serum off the slides before staining.
Incubate sections with primary antibody for at least 30 minutes at room temperature in a humid chamber, or overnight at 4°C. Wash 3 times in TBS.
Add enzyme-conjugated secondary antibody at the recommended dilution (see specific datasheet for details). Incubate for at least 30 minutes at room temperature. Wash 3 times in TBS.
Incubate with the appropriate substrate solution for the recommended period of time (it is suggested to useDAB substrate with HRP-conjugated antibodies, and Fast Red/Napthol AS-MX for Alkaline Phosphatase-conjugated antibodies). Wash once in water.
Counterstain with hematoxylin for 1-10 minutes. “Blue” with running water for 5 minutes. Then wash.
Mount in aqueous mounting medium, or alternatively dehydrate through a graded series of alcohols and xylene/solvent, and mount in synthetic mountant.

Notes
- Do not allow slides to dry out after the fixation step, as drying will result in damage to the tissue structure.
- Beware, certain substrates are soluble in alcohol – please refer to manufacturer's information for details.
- Appropriate controls should always be carried out. It may be useful to include a sample in which no primary antibody is used at all, in order to determine any non-specific binding of the secondary reagent to the target tissue.

In addition, you can also check the Abreviews section where 7 IHC-Fr reviews had been submitted by other customers:
http://www.abcam.com/CD68-antibody-FA-11-ab53444-reviews.html




I wish you the best of luck with your research.

Please let me know if there is anything else I can do for you.

I tried the PT given to me earlier but they didn't improve the results. Can I try ab31630 instead? 

ANSWER:

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

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 with ab31630 with the order number xxxxx.

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.

LOT NUMBER GR21055-1 + 962768 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM Non-specific staining, staining of capillaries SAMPLE mus musculus: heart, liver, spleen PRIMARY ANTIBODY ab53444, 1:30 till 1:300 in PBS-Tween 4°C on, 2h 37°C DETECTION METHOD flourescence POSITIVE AND NEGATIVE CONTROLS USED negative controls: sample without primary antibody ANTIBODY STORAGE CONDITIONS -20°C, aliquoted FIXATION OF SAMPLE Paraffin ANTIGEN RETRIEVAL citraconic anhydride 1h 97°C BLOCKING CONDITIONS none SECONDARY ANTIBODY anti rat igG Tritc (Dako) HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Change the antigen retrieval buffer (citric acid) change the lot of the primary antibody testing on frozen sections (liver, spleen)

ANSWER:

Vielen Dank für Ihre Email und dass Sie sich die Zeit genommen haben, unseren Fragebogen auszufüllen. Es tut mir leid, dass Sie Probleme mit unserem Antikörper hatten. Wir garantieren alle unsere Produkte so zu funktionieren wie auf dem Datenblatt beschrieben. Sollten wir daher die Ursache des Problems nicht finden können, werden wir Ihnen den Antikörper ersetzen (falls in den letzten 6 Monaten gekauft). Zuerst jedoch, möchte ich die folgenden Vorschläge zu Veränderung Ihres Protokolls machen und Ihnen auch die folgenden Fragen stellen, um das Problem besser verstehen zu können: 1.) Können Sie mir bitte ein Bild mit der Färbung in der Milz senden? Falls ich die Art der Färbung sehe, kann ich vielleicht besser die Ursache verstehen. 2.) Haben Sie eine Isotypen Kontrolle gemacht? Falls ja, können Sie mir bitte auch ein Bild senden. Falls nicht, würde ich sehr stark empfehlen eine zu machen, da so Hintergrund von dem sekundären Antikörper und dem Detektionssystem ausgeschlossen werden kann. 3.) Haben Sie auch eine positive Kontrolle des sekundären Antikörpers und des Detektionssystems? (Mit einem anderen primären Antikörper?) 4.) Können Sie mir sagen, ob Sie mit beiden Röhrchen und auf gefroren, sowie auch den Paraffin-Schnitten die gleichen Resultate erhalten? Ich freue mich auf Ihre Antworten um diesen Fall genauer untersuchen zu können.