Anti-CD68 antibody [ED1] (ab31630)

Reviews (18)Q&A (22)References (157)

Overview

  • Product name
    Anti-CD68 antibody [ED1]
    See all CD68 primary antibodies
  • Description
    Mouse monoclonal [ED1] to CD68
  • Host species
    Mouse
  • Specificity
    Ab31630 recognises a single chain glycoprotein of 110kD that is expressed predominantly on the lysosomal membrane of myeloid cells. Weak cell surface expression also occurs. The antigen is expressed by the majority of tissue macrophages and weakly by peripheral blood granulocytes. Studies have shown that the antigen recognised by ED1 is the rat homologue of human CD68.
  • Tested applications
    Suitable for: Flow Cyt, IHC-Fr, IP, WB, RIA, IHC-FrFl, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Tissue, cells or virus corresponding to CD68. Rat spleen cell.

  • Positive control
    • Flow Cyt: Rat peritoneal macrophages. IHC-P: Rat liver and kidney tissue.
  • General notes

    This product should be stored undiluted.

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 or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer
    pH: 7.40
    Preservative: 0.09% Sodium azide
    Constituent: PBS
  • Concentration information loading...
  • Purity
    Protein A purified
  • Purification notes
    Purified from tissue culture supernatant.
  • Clonality
    Monoclonal
  • Clone number
    ED1
  • Myeloma
    Sp2/0-Ag14
  • Isotype
    IgG1
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab31630 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
Flow Cyt 1/50 - 1/100.

Use 10µl of the suggested working dilution to label 106 cells in 100µl.
ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
IHC-Fr Use at an assay dependent concentration.

Fixation with acetone is recommended.
IP Use at an assay dependent concentration.
WB Use at an assay dependent concentration. Predicted molecular weight: 37 kDa.
RIA Use at an assay dependent concentration.
IHC-FrFl Use at an assay dependent concentration. PubMed: 25374513
IHC-P 1/100. Perform heat mediated antigen retrieval before commencing with IHC staining protocol. Alternatively protein digestion with trypsin or pronase prior to IHC protocol could be preformed.

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
    see all
  • 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

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD68 antibody [ED1] (ab31630)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD68 antibody [ED1] (ab31630)Chang et al PLoS One. 2017 Jun 20;12(6):e0179809. doi: 10.1371/journal.pone.0179809. eCollection 2017. Fig 3.

    Pulmonary immunochemical staining in rats with selective COX inhibition.

    CD68: SC-560 (Top left panel) diminished CD-68 positive macrophages (green arrow indicating brown cells) accumulation compared to control (Top right panel) and celecoxib (Bottom panel) groups (magnification 200x).

    Rat lung tissue were dissected free and fixed in 10% formalin solution.

    ab31630 used at a 1:200 dilution.

  • Immunohistochemistry (Frozen sections) - Anti-CD68 antibody [ED1] (ab31630)
    Immunohistochemistry (Frozen sections) - Anti-CD68 antibody [ED1] (ab31630)Miyata et al PLoS One. 2017 Apr 24;12(4):e0175421. doi: 10.1371/journal.pone.0175421. eCollection 2017. Fig 4.

    Effect of a pharmacological inhibition of a T cell function on IA progression.

    F344/Jcl rats were subjected to IA (intracranial aneurysms) induction and, at 14th day after the induction, slices from IA specimen induced at ACA-OA bifurcation were prepared for further analyses (IHC-Fr sections).

    Administration of Cyclosporine A was started 1 day before the induction and lasted for a whole experimental period (15 mg/kg, once a day). Systemic blood pressure was evaluated before sacrifice without any anesthesia.

    Number of infiltrated macrophages in IA lesions was evaluated after immunostaining for a macrophage marker CD68 (per 12,100 μm2). Data represents mean ± SEM. Representative images of immunostaining for CD68 (ab31630, green) and α-smooth muscle actin (α-SMA, red) and of nuclear staining DAPI (blue) are shown in lower left panels. Bar, 20 μm.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD68 antibody [ED1] (ab31630)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD68 antibody [ED1] (ab31630)Image courtesy of an Abreview submitted by Dr. Carl Hobbs, King`s College London, United Kingdom

    ab31630 staining CD68 in rat liver tissue sections by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections).

    Tissue was fixed with formaldehyde and a heat mediated antigen retrieval step was performed using citrate buffer pH 6. Samples were then blocked with 1% BSA for 10 minutes at 21°C followed by incubation with the primary antibody at a 1/800 dilution, for 2 hours at 21°C. A biotin-conjugated goat anti-mouse IgG polyclonal was used as secondary antibody at a 1/200 dilution.

    See Abreview

  • Flow Cytometry - Anti-CD68 antibody [ED1] (ab31630)
    Flow Cytometry - Anti-CD68 antibody [ED1] (ab31630)

    Staining of rat peritoneal macrophages with Alexa Fluor®647 conjugated mouse anti rat CD68, clone ED1 (ab31630, red filled histogram) following permeabilization with Leucoperm.

References

This product has been referenced in:
  • Santana-Codina N  et al. NCOA4 maintains murine erythropoiesis via cell autonomous and non-autonomous mechanisms. Haematologica N/A:N/A (2019). Read more (PubMed: 30630985) »
  • Liu W  et al. Mesenchymal stem cells alleviate the early brain injury of subarachnoid hemorrhage partly by suppression of Notch1-dependent neuroinflammation: involvement of Botch. J Neuroinflammation 16:8 (2019). Read more (PubMed: 30646897) »
See all 157 Publications for this product

Publishing research using ab31630? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-10 of 22 Q&A

Question

Dear Sir/Madam,

I am interested in using anti-CD68 antibody [ED1] (ab31630) in mouse brain paraffin embedded sections, but am wondering if it is necessary to use a mouse-on-mouse kit for this to work.
Kind regards

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Answer

Thank you for your enquiry regarding ab31630.
Since the sample and the host species of this primary antibody is the same (mouse), unfortunately there is a chance for getting high non-specific background signal.
You may be able to work around this problem if you apply ABC signal enhancing system which requires Biotin-conjugated secondary antibody.
If you need any further assistance in the future, please do not hesitate to contact me.

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Question

The description indicates for this CD68 antibody says that "Ab31630 recognises a single chain glycoprotein of 110kD," yet the predicted size is 37kDa. Is this correct? I am only detecting a 110kDa band in rat microglia by western blot. (I loaded 8ug/lane, 5% milk TBST 1hr block, ECL detection.)

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Answer

Thank you for your inquiry.

I can confirm that prediction of the 110 kDa is based on literature references as follows:
1. Bao, F. et al. (2004) Early anti-inflammatory treatment reduces lipid peroxidation and protein nitration after spinal cord injury in rats. J, Neuro-chem. 88:1335-1344.
2. Kim H, et al. Increased phosphorylation of caveolin-1 in the spinal cord of irradiated rats. J
Vet Sci. 2007 Dec;8(4):323-7. PubMed PMID: 17993744
3. http://www.ncbi.nlm.nih.gov/pubmed/7821959

These references show a band of 110kDa (ED1 is the rat homologue of human CD68). Looking at the available literature for Rat CD68 it appears that is accepted that it can appear on a Western blot across a range of 30-110kDa depending on conditions and experimental setup. This is most likely due to doublet and triplet formation although I have been unable to confirm this with referenced material.

I hope this information is helpful for you. Please do not hesitate to contact me again if you need more information.

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Question

-Previous lots of ab31630 and ab45977 worked very well but recent lots are not working.
- Observed white precipitates in vials

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Answer

Thank you for contacting us.

As discussed please send us the purchase order number. We will then check what went wrong with these antibodies.

Thanks!

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Question

Customer inquired about detecting presence of 7 different proteins in brain tissue via IHC-F. Would like 3-4 different host species for primary, secondary antibodies that do not overlap in spectra. Customer is also not sure if Caspase 3, Nogo, and neurofilament are necessary for detecting apoptosis / neuronal response.

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Answer

Thank you for contacting Abcam.

I managed to find antibodies against rat GFAP, FOX3, Iba1, and CD68 all raised in different hosts (chicken, rabbit, goat, and mouse, respectively). They each are highly rated by other researchers and have numerous specific published references. The list for these 4 antibodies is given below:
1. GFAP ab4674 (https://www.abcam.com/gfap-antibody-astrocyte-marker-ab4674.html)
2. FOX3 ab104225 (https://www.abcam.com/fox3neun-antibody-ab104225.html)
3. Iba1 ab5076 (https://www.abcam.com/iba1-antibody-ab5076.html)
4. CD68 ab31630 (https://www.abcam.com/cd68-antibody-ed1-ab31630.html)

I also compiled anti-chicken, rabbit, goat, and mouse secondaries with separate emission spectra. We don't have secondaries for these species that weren't raised in any of the 4 species conjugated to a fluorescent protein with a similar spectrum to DAPI. Thus, I think your best bet is to obtain a far-red filter (Cy5), that way you could still stain with DAPI as well. The donkey secondaries that I found with separate emission spectra are listed below:
1. anti-chicken: FITC (https://www.abcam.com/chicken-igy-secondary-antibody-ab63507.html)
2. anti-rabbit: DyLight 594 (Similar spectrum to Texas Red) (https://www.abcam.com/donkey-polyclonal-secondary-antibody-to-rabbit-igg-h-l-dylight-594-pre-adsorbed-ab96921.html)
3. anti-goat: DyLight 650 (Similar spectrum to Cy5) (https://www.abcam.com/donkey-polyclonal-secondary-antibody-to-goat-igg-h-l-dylight-650-pre-adsorbed-ab96938.html)
4. anti-mouse: DyLight 550 (similar spectrum to TRITC) (https://www.abcam.com/donkey-polyclonal-secondary-antibody-to-mouse-igg-h-l-dylight-550-ab96876.html)

The other antibodies you chose (Nogo, heavy neurofilament, caspase 3) seem to be good choices as well as they are tested in formaldehyde fixed, frozen sections via IHC in rat. Below are the antibodies we discussed against those three proteins:
1. anti-Nogo (https://www.abcam.com/nogo-antibody-ab32298.html)
2. anti-heavy neurofilament (https://www.abcam.com/200-kd-neurofilament-heavy-antibody-ab134459.html)
3. anti-caspase 3 (https://www.abcam.com/active-caspase-3-antibody-ab13847.html)

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

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Question

Dear Tech Support Team,

Please advise in regards to the following discrepancy in the datasheet:

in the specificity paragraph is written that Ab31630 recognizes a single chain glycoprotein of 110kD and the application section indicates " Predicted molecular weight: 37 kDa"

Thanks in advance for the clarification.

Kind Regards,

Read More
Answer

Thank you for your inquiry.
I can confirm that prediction of the 110 kDa is based on literature references as follows:
1. Bao, F. et al. (2004) Early anti-inflammatory treatment reduces lipid peroxidation and protein nitration after spinal cord injury in rats.
J, Neuro-chem. 88:1335-1344.
2. Kim H, et al. Increased phosphorylation of caveolin-1 in the spinal cord of irradiated rats. J
Vet Sci. 2007 Dec;8(4):323-7. PubMed PMID: 17993744
Both references show a band of 110kDa (ED1 is the rat homologue of human CD68). Looking at the available literature for Rat CD68 it appears that is accepted that it can appear on a Western blot across a range of 30-110kDa depending on conditions and experimental setup. This is most likely due to doublet and triplet formation although I have been unable to confirm this with referenced material.
I hope this information is helpful for you. Please do not hesitate to contact me again if you need more information.

Read More

Question


Thank you very much for your reply.
I did vary goat anti-mouse IgG concentration, 1:25, 1:50, 1:100. In
all cases, even without primary antibody, I do see signals in
controls. Here are the conditions used:
goat anti-mouse IgG: various conc
Primary: Mouse anti-CD68 [ED1] (Abcam ab31630) (or isotype or none)
Secondary: Rabbit anti-mouse IgG-biotin (Abcam ab98784)
HRP: Streptavidin-peroxidase polymer, ultrasensitive (Sigma S2438)
I am not 100% aware of what my supervisor has told you (as I joined
the team in the middle of this project and inherited the project from
a student. I myself have not tried FA-11 from Serotec in our tissue
of interest, kidney, but it did work for lung when I tried. Also, in
general, Abcam antibodies are of higher quality. So I have high hope
that FA-11 from Abcam can detect macrophage infiltration.

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Answer

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

Since the no primary control is showing staining, it's probably an issue with the secondary antibody. But I'll be happy to send you ab53444 as a free of charge replacement for the CD68 antibody. You'll have to use an anti-rat secondary antibody with ab53444 and I strongly encourage you to use one that is pre-adsorbed against mouse so you don't run into a similar problem.

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 of ab53444.

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.

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Question


To whom it may concern:

I am currently conducting the IHC experiments with your mouse anti-CD68 (ab31630) and goat anti-mouse F(ab) (ab6668)

I followed your protocol found that it was not still working. As you can see on the top right of the attached figure, we see signals in isotype control; this either indicates that blocking by goat anti-mouse F(ab) is not working, or that secondary antibody (ab98784) has non-specific binding.

This sample is high fat diet fed mouse liver tissue. These tissues are taken from identical animal. We expect the similar picture with anti-Cd68 with the one with anti-F4/80 (bottom left panel), as both CD68 and F4/80 are macrophage specific markers.

So in this system, even with the help of goat anti-mouse F(ab), this anti-CD68 does not detect the macrophage we hoped to see. I would really appreciate it if you could replace it with rat anti-CD68 monoclonal [FA-11], as this is supposed to react with mouse, and applicable to IHC-P, and without the need for goat anti-mouse.

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Answer

Thank you for your inquiry. I am sorry to hear that you are still not seeing optimal staining results with this antibody.

My colleague Jackie had sent you the goat anti-mouse F(ab) ab6668 free of charge to help with the blocking experiment. What concentration are you using? It may be that you have to increase the concentration of this block and incubate it overnight at 4C as the block doesn't seem complete.

Have you run a no-primary control?

Your colleague had said in a previous correspondence that you couldn't get the FA-11 clone working from AbDSerotec. Is there a reason why you want to try this same clone again if it hadn't worked previously?

I look forward to your reply.

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Question

it was for sure the order issued on February the 15th.
thanks!
Libero

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Answer

Thank you for kindly confirming these details.

As requested, we would be pleased to arrange this free of charge replacement. In order to do this, I am forwarding these details on to our distributors team who will be happy to organize this for you. This will be sent via Prodotti Gianni andI have copied them into this email so they are aware of this case.

Original order number: ######

Original purchase 1 X vial ab31630
Please provide an alternative FOCR vial ofab125212 for the customer

Thank you for your help and cooperation with this case. Please do not hesitate to contact us if you need anything further.

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Question

Tthank you very much for the info & assistance; amongst the three possibilities that you suggested, both ab76308 and ab125212 look like they'd be good choices. The latter would be more versatile, not only wb but also IHC-Fr, but I am puzzled by the specification of the minimum amount per lane in the datasheet, which is a piece of information that I am not used to see. Does it mean that ab125212 is somewhat less sentitive thanab76308? If so, I'd definitely go for ab76308.

Once we decide for one of the two, how do we proceed to arrange the replacement?

Best,

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Answer

Thank you for your reply.

The information regarding western blot on the datasheet for ab125212 is provided as the detection limit in western blot for this particular antibody is known to be 0.1ng/lane. For other antibodies, the detection limit is not always assessed and so this information would not be on the datasheet.

Therefore, we do not know the detection limit of ab76308 and this is not on the datasheet and so we cannot compare this to ab125212 in this way and cannot say which is the most sensitive.

Although we produce many in-house antibodies, we obtain other antibodies from a wide range of sources. Therefore, comparison experiments will not often have been done. However, I can reassure you that Abcam antibodies are all guaranteed in the applications and species listed on the datasheet.

To proceed with the free of charge replacement, please let me know which antibody you would like to receive and I can arrange it all for you via PodottiGianni.

I look forward to hearing from you. Should you have any further questions please do not hesitate to ask.

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Question

Hello Kate,
we did perform the last test, and I am afraid that the results did not change from what we had already seen.
Here are the details of the experiment:
30µg of total protein per sample were heat-denatured in the presence of loading buffer and reducing reagent, in a 4-12% NuPage gel.
The resulting nitrocellulose filter contained two lanes loaded with whole muscle lysate, prepared from murine muscle pre-damaged with bupivacaine and harvested three days post damage (it is well-established that at this stage most of the inflammatory cell population is comprised of M1 macrophages, which are CD68 positive); two lanes loaded with total cell lysate prepared from two murine macrophage cell lines (raw and J774), one lane loaded with total cell lysate from murine myoblasts C2C12.
We performed an overnight saturation with TBS-T milk5% at 4 degrees, followed by one hour incubation with primary ab (1:250 in saturating solution), 3 washes in TBST, one hour incubation with secondary ab (1:2000 in saturating solution).
As you can see from the image, in the 37kD range (where the band should be) there is no clear signal present in the muscles or in the cultured macrophages and absent in the C2C12.

At this point we can safely assume that this particular ab does not work for us; we absolutely need to quantify the M1 macrophages in our samples and I was wondering if you could suggest any other alternative from your catalog, which could then be sent to us as a replacement instead of issuing a refund.

thank you in advance for your assistance,

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Answer

Thank you for your reply and for kindly confirming these details.

I would like to reassure you that we monitor feedback closely on a weekly basis and we are not currently concerned about the general quality of this antibody or this batch. Regrettably, I can suggest you have received a bad vial on this occasion.

Therefore, we would be pleased to provide a replacement from the same antibody, which will still be covered by our guarantee. Alternatively, I fully understand your concerns and if you prefer an alternative replacement or credit note in this case, I will be pleased to arrange this for you.

For alternatives, we have the following CD68 antibodies tested in mouse and western blot (please note these are not mouse monoclonal antibodies):

ab53444 Anti-CD68 antibody [FA-11]
Rat monoclonal
Flow Cyt, IHC-Fr, IHC-P, IP, WB
Reacts with: Mouse
https://www.abcam.com/index.html?datasheet=53444 (or use the following: https://www.abcam.com/index.html?datasheet=53444).

ab125212 Anti-CD68 antibody
Rabbit polyclonal
IHC-Fr, IHC-P, WB
Reacts with: Mouse, Rat
https://www.abcam.com/index.html?datasheet=125212 (or use the following: https://www.abcam.com/index.html?datasheet=125212).

ab76308 Anti-CD68 antibody [EPR1392Y]
Rabbit monoclonal
WB
Reacts with: Human, Mouse, Rat
https://www.abcam.com/index.html?datasheet=76308 (or use the following: https://www.abcam.com/index.html?datasheet=76308).

I hope this will be helpful. I look forward to hearing from you with details of how you would like to proceed.

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1-10 of 22 Q&A

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