Overview

  • Product name

  • Description

    Rabbit polyclonal to CD68
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IHC-Pmore details
  • Species reactivity

    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide within Human CD68 aa 300 to the C-terminus (C terminal). The exact sequence is proprietary.
    Database link: P34810

  • Positive control

    • Human tonsil tissue; Raji and SH SY5Y cell lysates.
  • General notes

    The user must validate any other storage conditions.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C. Do Not Freeze.
  • Storage buffer

    pH: 7.6
    Preservative: 0.1% Sodium azide
    Constituents: PBS, 1% BSA
  • Concentration information loading...
  • Purity

    Immunogen affinity purified
  • Clonality

    Polyclonal
  • Isotype

    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab125047 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
WB 1/50. Predicted molecular weight: 37 kDa.
IHC-P 1/100. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

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

  • ab125047 staining CD68 in Mouse vasculature tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 10% serum for 5 minutes at 20°C; antigen retrieval was by heat mediation in a tris buffer. Samples were incubated with primary antibody (1/100 in antibody diluent) for 16 hours at 4°C. An Alexa Fluor® 488-conjugated Goat anti-rabbit IgG polyclonal (1/200) was used as the secondary antibody.

    See Abreview

  • Anti-CD68 antibody (ab125047) at 1/50 dilution + SH SY5Y cell line lysate

    Predicted band size: 37 kDa

  • ab125047, at 1/100, staining CD68 in formalin fixed, paraffin embedded Human tonsil tissue by Immunohistochemistry.

References

This product has been referenced in:

  • Mori D  et al. Pioglitazone strengthen therapeutic effect of adipose-derived regenerative cells against ischemic cardiomyopathy through enhanced expression of adiponectin and modulation of macrophage phenotype. Cardiovasc Diabetol 18:39 (2019). Read more (PubMed: 30902059) »
  • Liu L  et al. Anti-spike IgG causes severe acute lung injury by skewing macrophage responses during acute SARS-CoV infection. JCI Insight 4:N/A (2019). Read more (PubMed: 30830861) »
See all 11 Publications for this product

Customer reviews and Q&As

1-10 of 11 Abreviews or Q&A

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (human ovarian cystedenocarcinoma tissue sections)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: bicarbonate buffer pH 9.0
Specification
human ovarian cystedenocarcinoma tissue sections
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
Fixative
Formaldehyde

Dr. Arpita Kulshrestha

Verified customer

Submitted Aug 13 2015

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (mouse breast tumor)
Permeabilization
Yes - Citrate buffer pH 6.0
Specification
mouse breast tumor
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
Fixative
OCT

Dr. Arpita Kulshrestha

Verified customer

Submitted Jul 17 2015

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 25% · Temperature: 25°C
Antigen retrieval step
None
Sample
Sheep Tissue sections (Lymph Node)
Specification
Lymph Node
Permeabilization
No
Fixative
Zinc Salts

Helen Todd

Verified customer

Submitted Aug 20 2014

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 25% · Temperature: 25°C
Antigen retrieval step
None
Sample
Cow Tissue sections (Mesenteric Lymph Node)
Specification
Mesenteric Lymph Node
Permeabilization
No
Fixative
Zinc Salts

Helen Todd

Verified customer

Submitted Aug 18 2014

Application
Western blot
Loading amount
20 µg
Gel Running Conditions
Reduced Denaturing
Sample
Human Tissue lysate - whole (Skeletal Muscle (Gastrocnemius))
Specification
Skeletal Muscle (Gastrocnemius)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Nov 21 2013

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step
Serum as blocking agent for 5 minute(s) · Concentration: 10% · Temperature: 20°C
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Tris
Sample
Mouse Tissue sections (Mouse Vasculature)
Specification
Mouse Vasculature
Permeabilization
No
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Sep 19 2013

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step
Serum as blocking agent for 5 minute(s) · Concentration: 10% · Temperature: 25°C
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Tris (pH8) Cit (pH) does not work
Sample
Human Tissue sections (Skeletal Muscle (Gastrocnemius))
Specification
Skeletal Muscle (Gastrocnemius)
Permeabilization
No
Fixative
Methacarn

Abcam user community

Verified customer

Submitted Sep 17 2013

Question

A customer has enquired about the following product: https://www.abcam.com/cd68-antibody-ab125047.html Anti-CD68 antibody



‘We recently purchased an Abcam anti-CD68 rabbit polyclonal antibody (ab125047) which uses a peptide from the C-terminus of CD68 as the immunogen and claims to produce a 37 kDa band (predicted MW, so unglycosylated) on western blot. We have compared the results with those obtained using a Dako anti-CD68 (KP1 clone) mouse monoclonal (M 0814), which uses the lysosomal fraction from alveolar macrophages as the immunogen, and claims to target a 110 kDa band on western blot which corresponds to the glycosylated form of the protein.



In our hands, the two antibodies do not appear to stain the same population of cells in FFPE sections of human brain, and the cells do not necessarily colocalise using immunofluorescence. I have attached some 60x images of a co-localisation experiment in which mouse CD68 is seen in red (594) and Abcam rabbit CD68 is seen in green (488). The mouse antibody appears to localise with lysosomes, whereas the rabbit antibody labels whole cells.



We are not very happy about this because we need to colocalise CD68 with other mouse antibodies. Your assistance would be appreciated.’



The images and technical response form are attached.





Antibody Code: ab125047
Batch Number: GR87304-1

GENERAL INFORMATION

Antibody Storage Conditions (temperature/reconstitution etc)
fridge (2-8 C)


Description of the Problem (high background, low signal, non-specific staining etc)
antibody does not label same cells as Dako CD68 (KP1 clone) mouse monoclonal antibody, and the pattern of staining in the tissue is different


Sample (Species/Tissue/Cell Type/Cell Line etc)
postmortem human brain


Fixation of Sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration)
formalin-fixed paraffin embedded (typically 2 weeks fixation)


Antigen Retrieval (Enzymatic method, Heat mediated technique etc)
120 C for 5 - 10 min followed by 95 C for 40 min in decloaking chamber, in Tris 10 mM / EDTA 1 mM buffer (pH 9)


Permeabilization step
0.1% Tween-20 in wash buffers (PBST), blocking serum, and antibody diluent


Blocking Conditions (Buffer/time period, Blocking agent)
10% normal goat serum in PBST for 20 min, Sudan Black in 70% alcohol for 4 min to reduce autofluorescence


Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, wash step)
cocktail containing Abcam anti-CD68 (ab125047) rabbit polyclonal, and Dako anti-CD68 (KP1 clone) mouse monoclonal (m 0814), both at 1:50 dilution for 1 hour at room temperature


Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, wash step)
on different sections, either cocktail containing Alexafluor anti-mouse raised in goat (488), and Alexafluor anti-rabbit raised in goat (568), or Alexafluor anti-mouse raised in goat (594), and Alexafluor anti-rabbit raised in goat (488)


Detection Method
immunofluorescence


Positive and negative controls used (please specify)
no negative control, but these sections have previously been shown to contain CD68-positive microglia


OPTIMIZATION ATTEMPTS (PROBLEM SOLVING)

How many times have you tried the IHC?
twice with fluorescence, but also several times with brightfield and DAB; another person has tried IF also

Have you run a “No Primary” control? Not yet (Delete one)

Do you obtain the same results every time? Yes (Delete one)

What steps have you altered?
antigen retrieval method, antibody concentrations, incubation times

Additional Notes
I tried IF double labelling, and Abcam CD68 labels cell body and processes beautifully, whereas Dako CD68 labels lysosomes, but not necessarily on the same cells

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 from ab125047 Anti-CD68 antibody. I would also appreciate if you can confirm some further details:

1. I would first try to establish these two antibodies independent from each other us it is very likely that they need different protocol optimization steps.

2. Brain tissue is usually very delicate to handle, and the antigen retrieval perfomed is very harsh, which may lead to wrong staining. I would like to suggest to switch to a different method completely (like microwave or steamer) or to reduce the time down to a maximum of 25 min in total (1-5 min maximum for 120C) and to perform a time series to find the optimal retrieval point.

3. Blocking the autoflourescence with Sudan Black is a good idea. However, I would suggest to postpone this step after the secondary antibody has been incubated to ensure that the immunostaining is completed (and that the protective block protein layer stays in place). Please also notice that this kind of quenching is not sufficient enough for FITC (or 488) and Alexa 594 as it may not reduce the emission signal noticeably. Please see attachment.


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 your inquiry.

I agree with your customer that there is not much difference with both antibodies and that we do not have any customer feedback for them yet.

I can confirm that both are covered by our guarantee for IHC-P and human samples. I would suggest to use a positive control with the brain samples. For example human tonsil.

Please also suggest to your customer to write an Abreview. This will be valuable information for other researchers and your customer could earn Abpoints that can be redeemed as discount on future purchases or Amazon vouchers.

I am sorry I do have more information for you and hope this is nevertheless helpful.

Read More

Question
Answer

Thank you for contacting us.

The IgG concentration of our current lot of ab125047, lot GR87304-2, is 0.3 mg/ml.

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

Read More

1-10 of 11 Abreviews or Q&A

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