Overview

  • Product name

    Anti-Macrophage antibody [RM0029-11H3]
    See all Macrophage primary antibodies
  • Description

    Rat monoclonal [RM0029-11H3] to Macrophage
  • Host species

    Rat
  • Tested applications

    Suitable for: ICC/IF, IP, Flow Cyt, IHC-Pmore details
  • Species reactivity

    Reacts with: Mouse
  • Immunogen

    Isolated mouse peritoneal macrophages

  • Positive control

    • Spleen, Lymph node, Diseased (GBM) mouse kidney tissue. IF/ICC: RAW246.7

Properties

Applications

Our Abpromise guarantee covers the use of ab56297 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 10 µg/ml.
IP 1/100.
Flow Cyt Use 2µg for 106 cells.

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

 

IHC-P Use at an assay dependent concentration. Perform enzymatic antigen retrieval before commencing with IHC staining protocol.

Target

  • Relevance

    Macrophages comprise of many forms of mononuclear phagocytes found in tissues. Mononuclear phagocytes arise from hematopoietic stem cells in the bone marrow. After passing through the monoblast and promonocyte states of the monocyte stage, they enter the blood, where they circulate for about 40 hours. They then enter tissues and increase in size, phagocytic activity, and lysosomal enzyme content becomming macrophages. Among the functions of macrophages are nonspecific phagocytosis and pinocytosis, specific phagocytosis of opsonized microorganisms mediated by Fc receptors and complement receptors, killing of ingested microorganisms, digestion and presentation of antigens to T and B lymphocytes, and secretion of a large number of diverse products, including many enzymes including lysozyme and collagenases, several complement components and coagulation factors, some prostaglandins and leukotrienes, and many regulatory molecules (Interferon, Interleukin 1). Among cells that are now recognised as macrophages are histiocytes, Kupffer cells, osteoclasts, microglial cells, synovial type A cells, interdigitating cells, and Langerhans cells (in normal tissues) and epithelioid cells and Langerhans-type and foreign-body-type multinucleated giant cells (in inflamed tissues).
  • Alternative names

    • macrophages antibody

Images

  • ICC/IF image of ab56297 stained RAW246.7 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab56297, 10µg/ml) overnight at +4°C. The secondary antibody (green) was ab96887, DyLight® 488 goat anti-rat IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM

  • Bouin’s solution fixed and paraffin embedded mouse kidney section from anti-GBM model was subjected to immunohistochemistry staining (ABC) of Macrophage using ab56297.
  • Ab56297 staining macrophage in mouse brain tumour tissue sections by (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and blocked with 0.5% TNB blocking reagent for 30 minutes at 25°C. Samples were incubated with primary antibody at 1/100 dilution for 18 hours at 4 C. A goat anti-rat IgG H&L (HRP) (ab7097) was used at 1/500 dilution.

    See Abreview

  • Overlay histogram showing RAW 264.7 cells stained with ab56297 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab56297, 2µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rat IgG (H+L) (ab98386) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rat IgG2a, kappa monoclonal [aRTK2758] (ab18450, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in RAW 264.7 cells fixed with 80% methanol/permeabilized in 0.1% PBS-Tween used under the same conditions.

  • Immunohistochemical analysis of murine tumour tissue, staining Macrophage with ab56297. Antigen retrieval was performed under high pressure in 10 mM EDTA buffer (pH 8.0) before incubation with primary antibody.

References

This product has been referenced in:

  • Zhao W  et al. Arbutin attenuates hydrogen peroxide-induced oxidative injury through regulation of microRNA-29a in retinal ganglion cells. Biomed Pharmacother 112:108729 (2019). Read more (PubMed: 30970524) »
  • Baudoux T  et al. CD4+ and CD8+ T Cells Exert Regulatory Properties During Experimental Acute Aristolochic Acid Nephropathy. Sci Rep 8:5334 (2018). IHC-P ; Mouse . Read more (PubMed: 29593222) »
See all 21 Publications for this product

Customer reviews and Q&As

1-7 of 7 Abreviews or Q&A

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Normal Liver)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate pH 6.0
Permeabilization
Yes - triton-X
Specification
Normal Liver
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Sep 06 2018

Answer

Thank you for your reply.

I will contact * and determine the best way to get the FOCR sent to you as quickly as possible. Once we have sorted the situation out, we will get back to you and let you know when you should have the free of charge replacement.

Thank you for your patience in this matter.

Read More

Answer

Thank you for your reply.

I would be happy to credit the cost of the antibody back to you, or I could send one of the antibodies that the customer would like to try as a free of charge replacement instead.

Please let me know how you would like to continue and if you could also provide me either the Abcam order number or the PO number that was used to purchase ab56297, then I would be happy to get this issue resolved for you.

I look forward to your reply.

Read More

Question

A costumers is having problems with:



Rat Monoclonal [RM0029-11H3] to Macrophage

ABCAM - Code ab56297

Batch: ******



Vial received at the lab on Nov 08, 2011.

Storage: 2-8oC, original vial since arrival at the lab





He is using IHC-P technique and says that the antibody isn't working.

It's negative on all tested mouse tissues (mouse ear epithelium with macrophage presence identified by HE staining).



Protocol tested with ab56297:



Dewax

Xilol (3 X 10 minutes)

Acetone (1 X 10 minutes)



Rehydrate

Baths on decreasing grades of Ethanol (96%, 70%, 50% and 30%)

Washes in distilled water and PBS/ Tween 0,1% (pH 7,2-7,4)

Antigen Retrieval

Water Bath (95-99oC, for 30 minutes)

Tested Retrieval Solutions: Citrate, pH 6,0 and Tris/EDTA pH 9,0

Cooling at room temperature for 15-20 minutes

Washes in distilled water and PBS/ Tween 0,1% (pH 7,2-7,4)



Peroxidase Blocking

H2O2 3%, 30 minutes at room temperature, in the dark

Washes in PBS/ Tween 0,1% (pH 7,2-7,4)



Protein Blocking

Milk, 40 minutes at room temperature



Primary Antibody

Tested Dilutions: 1/10, 1/25, 1/50,1/100 and 1/200

Incubation: Overnight, 2-8oC

Washes in PBS/ Tween 0,1% (pH 7,2-7,4)



Secundary Antibody

Biotinilated Anti-Rat IgG (Vector), diluted 1:500

Incubation: 40 minutes, at room temperature

Washes in PBS/ Tween 0,1% (pH 7,2-7,4)



Streptoavidin-HRP ComplexWashes in distilled water.



Counterstaining with Hematoxilin, dehydration and slide mounting







Streptoavidin-HRP Kit (Dako)

Incubation: 30 minutes, at room temperature

Washes in PBS/ Tween 0,1% (pH 7,2-7,4)

Chromogen

Liquid DAB (DAKO)

Incubation: 30 seconds, at room temperature

What do you think about that case?


1-) Could this be a problem with the reagent vial (ab56297 - Batch GR49874-1)?

2-) If you think the antibody is OK, can you give any clue to improve protocol?



I'm forward to hearing from you.

Thanks for your attention.

Read More
Answer

Thank you for contacting Abcam.

The antibody is covered under our Abpromise for six months and is guaranteed to work in IHC-P on mouse samples . If we cannot resolve the issue you are having with the antibody then I would be happy to either send a replacement antibody or to process a refund.

I just had one further question for your customer: Have they tried using the antibody without any antigen retrieval methods? I ask, as we have some Abreviews and publications that show the antigen retrieval step is not necessary. Therefore omitting this step may prove to be successful.

As mentioned above, if we cannot resolve the issue you are having, then we would be happy to replace or refund the antibody for the customer.

I look forward to your reply.

Read More

Answer

Thank you for contacting us. We have not tested this product to see whether it cross-reacts with monocytes.

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

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

Read More

Answer

We are happy to help customers find the most suitable products for their research purposes; however we are not specialized in this particular field and do not want to recommend a product that may be unsuitable. We encourage customers to consult the latest literature available through PubMed and other resources in order to find the most up-to-date information about their specific research interests. I am sorry to confirm that I do not know of an antibody that will recognize Macrophages and Eosinphils specifically and at the same time. I would like to recommend to review the following antibodies to stain for macrophages: ab6640 (Anti-F4/80 antibody [CI:A3-1]): Click here (or use the following: https://www.abcam.com/index.html?datasheet=6640). ab56297 (Anti-Macrophage antibody [RM0029-11H3]): Click here (or use the following: https://www.abcam.com/index.html?datasheet=56297). Both these antibodies are tested and guaranteed for IHC-P in mouse samples. Maybe it would be suitable to use eosin to stain for eosinophils? Eosin stains the cytoplasmic granules of eosinophils strongly. I am sorry that I could not be more helpful, but I hope that the available literature in this area can provide some clarification. Please do not hesitate to contact us again with other needs or with any questions about our products.

Read More
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (brain tumor)
Specification
brain tumor
Fixative
Paraformaldehyde
Antigen retrieval step
None
Permeabilization
No
Blocking step
(agent) for 30 minute(s) · Concentration: 0.5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Apr 22 2009

Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
For licensing inquiries, please contact partnerships@abcam.com

Sign up