Overview

  • Product name
    Anti-CD68 antibody [FA-11]
    See all CD68 primary antibodies
  • Description
    Rat monoclonal [FA-11] to CD68
  • Host species
    Rat
  • Specificity
    ab53444 detects surface CD68 at low levels in resident mouse peritoneal macrophages which can be enhanced with thioglycollate stimulation.
  • Tested applications
    Suitable for: ICC/IF, WB, IP, IHC-Fr, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse
  • Immunogen

    Tissue, cells or virus corresponding to Mouse CD68.

  • Positive control
    • IHC-Fr: Mouse lung, spleen and heart tissue sections; ICC/IF: RAW 246.7 cell line. Flow Cyt: Mouse peritoneal macrophages.
  • General notes

    Although some customers have had success with this antibody in IHC-P, we are unable to obtain positive results in this application and so cannot recommend it for IHC-P. We batch test the antibody in IHC-Fr.

    This antibody clone is manufactured by Abcam.

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term.
  • Storage buffer
    pH: 7.40
    Preservative: 0.02% Sodium azide
    Constituents: PBS, 6.97% L-Arginine
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Purification notes
    Purified IgG prepared by affinity chromatography on Protein G from tissue culture supernatant.
  • Clonality
    Monoclonal
  • Clone number
    FA-11
  • Isotype
    IgG2a
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab53444 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ICC/IF Use a concentration of 1 µg/ml.
WB Use at an assay dependent concentration. Predicted molecular weight: 36 kDa.
IP Use at an assay dependent concentration.
IHC-Fr Use a concentration of 0.1 - 5 µg/ml.
Flow Cyt 1/50 - 1/100.

Use 10µl of the suggested working dilution to label 106 cells in 100µl. Membrane permeabilisation is required for this application.

ab18450 - Rat monoclonal IgG2a, is suitable for use as an isotype control with this antibody.

Target

  • Function
    Could play a role in phagocytic activities of tissue macrophages, both in intracellular lysosomal metabolism and extracellular cell-cell and cell-pathogen interactions. Binds to tissue- and organ-specific lectins or selectins, allowing homing of macrophage subsets to particular sites. Rapid recirculation of CD68 from endosomes and lysosomes to the plasma membrane may allow macrophages to crawl over selectin-bearing substrates or other cells.
  • Tissue specificity
    Highly expressed by blood monocytes and tissue macrophages. Also expressed in lymphocytes, fibroblasts and endothelial cells. Expressed in many tumor cell lines which could allow them to attach to selectins on vascular endothelium, facilitating their dissemination to secondary sites.
  • Sequence similarities
    Belongs to the LAMP family.
  • Post-translational
    modifications
    N- and O-glycosylated.
  • Cellular localization
    Cell membrane and Endosome membrane. Lysosome membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • CD 68 antibody
    • CD68 antibody
    • CD68 antigen antibody
    • CD68 molecule antibody
    • CD68_HUMAN antibody
    • DKFZp686M18236 antibody
    • gp11 antibody
    • Gp110 antibody
    • LAMP4 antibody
    • Macrophage antigen CD68 (microsialin) antibody
    • MACROPHAGE ANTIGEN CD68 antibody
    • Macrosialin antibody
    • SCARD1 antibody
    • Scavenger receptor class D member 1 antibody
    see all

Images

  • Dual fluorescence combining IL-6 with markers for macrophages (CD68).

    O, P, and Q, dual labeling of IL-6 (red) and marker of macrophage (green) in db/db mouse heart tissues. Arrows in Q show the specific CD68 staining with absence of IL-6 staining. (R and S) Negative control: arrows show the absence of staining in vessels with control IgG and without primary antibodies. (T) Staining of nuclei with DAPI (blue) in heart tissues of the db/db mice.

    To identify and localize IL-6 protein in coronary arterioles, transverse sections of the mouse heart were stained using markers of endothelial cells, vascular smooth muscle cells, and macrophages. Freshly isolated hearts were embedded and frozen in OCT and sectioned at 5 μm. Slides were incubated with blocking solution (10% donkey serum in PBS) and permeabilized (0.1% Triton X-100 in PBS). Primary antibodies to IL-6 (goat polyclonal 15 micro g/ml, AF-406-NA; R&D) or macrophage marker CD68 (rat monoclonal, 1:1000, ab53444; Abcam) were used for sequential double immunofluorescence staining. Secondary antibodies were conjugated with the fluorophores FITC or Texas red. Sections were mounted in an anti-fading agent (Slowfade gold with DAPI; Invitrogen), and then the slides were observed and analyzed with a fluorescence microscope (IX81; Olympus) with a x40 objective (0.90 numerical aperture). For negative controls, primary antibodies were replaced with goat polyclonal IgG (Abcam), rabbit polyclonal IgG (GeneTex), and rat monoclonal IgG (Abcam) isotype controls at the same concentration. The specificity of the primary antibody was confirmed as the absence of immunofluorescence staining signals in the IL-6-/- mice.

    Diabetic mice (db/db).

     

  • Formaldehyde-fixed, frozen mouse lung tissue sections stained for CD68 using ab53444 at a 1/250 dilution in immunohistochemical analysis. Tissue sections were blocked using 1% BSA as a blocking agent for 10 minutes at 21°C. Primary antibody was incubated for 2 hours at 21°C. Secondary antibody was a biotin-conjugated goat anti-rat IgG at 1/250 dilution.

    See Abreview

  • IHC image of CD68 staining in mouse lung frozen tissue section. The section was incubated with ab53444, 0.1µg/ml, overnight at 4C. A goat anti-Rat biotinylated secondary antibody was used to detect the primary, and visualized using an ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

  • ab53444 staining CD68 in mouse spleen tissue by Immunohistochemistry (Frozen sections). Antibody was detected with HRP-conjugated Goat anti-Rat IgG, showing staining in the red pulp.
  • ab53444 stained in RAW 246.7 cells. Cells were fixed with 100% methanol (5 min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% Tween for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab53444 at 1µg/ml and ab7291 (Mouse monoclonal to alpha Tubulin - Loading Control) used at a 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150165, Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed (pseudo-colored green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594), preadsorbed (colored red), both used at 1/1000 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1hour at room temperature.

  • Staining of permeabilised mouse peritoneal macrophages with Rat anti mouse CD68 (ab53444).
  • ab53444 staining mouse heart tissue by Immunohistochemistry (IHC-frozen sections). Tissue underwent fixation in paraformaldehyde, no permeabilization and blocked in 1% serum for 10 minutes at 20°C. The primary antibody used undiluted and incubated with sample for 16 hours at 20°C. A Biotin conjugated goat polyclonal to rat Ig, diluted 1/100 was used as the secondary.  

    See Abreview

  • ab53444 at 1/100 dilution staining CD68 in mouse spleen tissue by immunohistochemistry (frozen sections). Sections were acetone fixed prior to blocking in 8% milk for 40 minutes at 36°C and then incubated with ab53444 for 20 hours at 4°C. A biotin conjugated goat polyclonal to rat Ig, diluted 1/400, was used as the secondary antibody.

    See Abreview

  • ab53444 staining CD68 in murine heart tissue by Immunohistochemistry (Frozen sections).
    Tissue was fixed in acetone, permeabilized using 0.3% Triton, blocked with 10% serum for 30 minutes at 20°C, then incubated with ab53444 at a 1/1000 dilution for 16 hours at 4°C. The secondary used was a FITC conjugated goat anti-rat polyclonal, used at a 1/1000 dilution.

    See Abreview

References

This product has been referenced in:
  • Bolós M  et al. Maturation Dynamics of the Axon Initial Segment (AIS) of Newborn Dentate Granule Cells in Young Adult C57BL/6J Mice. J Neurosci 39:1605-1620 (2019). Read more (PubMed: 30651327) »
  • Wang Z  et al. Decellularized neonatal cardiac extracellular matrix prevents widespread ventricular remodeling in adult mammals after myocardial infarction. Acta Biomater 87:140-151 (2019). Read more (PubMed: 30710713) »
See all 126 Publications for this product

Customer reviews and Q&As

11-20 of 27 Abreviews or Q&A

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Lung)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: pH6 (Ventana mRcc)
Permeabilization
No
Specification
Lung
Fixative
10% Neutral Buffered Formalin

Abcam user community

Verified customer

Submitted Oct 18 2012

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 ab125212.

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.

Read More

Question
Answer

Thank you for contacting us.

Your credit note ID is XXXXXX.

I am sorry that this antibody did not perform as stated on the datasheet, I have asked our Finance department to issue a credit note for you. The credit note may be used in one of the following ways:

(1) Redeemed against the original invoice if this hasn't already been paid.
(2) Held on the account for use against a future order.
(3) A full refund can be offered where no other invoices are outstanding.

Please contact your Finance department to confirm how you would like the credit note to be used and ensure it is not redeemed without your knowledge.

To specifically receive a refund please ask your Finance department to contact our Finance department at creditcontrol@abcam.com or by telephone using the information at the “Contact Us” link in the top right corner of our website.

The credit note ID is for your reference only, please refer to the credit note ID in any correspondence with our accounting department. We will send you the completed credit note by email or postal mail with the actual credit note number which will start with the letters CGB.

I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service should you require further expert advice

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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.

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Question


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.

Read More
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.

Read More

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:
https://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.

Read More

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.

Read More
Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (skin)
Specification
skin
Fixative
Acetone
Permeabilization
No
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Dec 05 2011

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (heart)
Specification
heart
Fixative
Acetone
Permeabilization
Yes - 0.3 % Triton
Blocking step
Serum as blocking agent for 30 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Nov 01 2011

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.

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11-20 of 27 Abreviews or Q&A

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