CD68

Hi
can anyone provide me the information (articles) about reactivity of monoclonal CD11b antibody clone CA16.3E10 with bovine protein

According to AbD Serotec, this antibody reacts with the protein from goat, cat, mustelid (weasel), pig, and bovine. link

Hi
can anyone provide me the information (articles) about reactivity of monoclonal CD11b antibody clone CA16.3E10 with bovine protein

Thank you very much indeed for your informative reply. I would like to do CD14, CD11b&c IHC for localizing ATMs and have already done CD68 IHC for ATMs. But granulocytes are also positive for CD14, CD11b&c. In that case, could you please tell me how I can differentiate the granulocytes from ATMs?

Here are a couple links to pdfs with tons of information on the differentiation of hematopoietic cells via surface markers: link, link

Here's a link to a company that cells reagents specifically for this area.

Thank you very much indeed for your informative reply. I would like to do CD14, CD11b&c IHC for localizing ATMs and have already done CD68 IHC for ATMs. But granulocytes are also positive for CD14, CD11b&c. In that case, could you please tell me how I can differentiate the granulocytes from ATMs?

Hi
Good day. Can anyone of you let me know whether CD68-negative cells might expess other call surface markers for adipose tissue macrophages such as CD11b, CD11c, CD14 etc. or only CD68-positve cells in adipose tissues can express one, two or all of them. It would be very helpful for me if I have your idea(s). Really I don't know whether this issue is relating directly to this subject or threat, sorry for that. Thanks in advance.
best regards

Since CD68 is the main macrophage cell surface marker, I'm not sure what you are looking at if they are CD68-negative. As you know, the entire cell surface marker issue is very complex. Granulocytes are CD68-neg but positive for CD14, CD11b&c. Monocytes and macrophages have CD68, CD14, CD11 and a bunch of other markers. So, CD68-neg cells can certainly express the other markers you mention but they won't be ATMs, per se.

Here are a couple papers that talk about the issue of cell surface markers on adipose tissue macs:
link
link

Hi
Good day. Can anyone of you let me know whether CD68-negative cells might expess other call surface markers for adipose tissue macrophages such as CD11b, CD11c, CD14 etc. or only CD68-positve cells in adipose tissues can express one, two or all of them. It would be very helpful for me if I have your idea(s). Really I don't know whether this issue is relating directly to this subject or threat, sorry for that. Thanks in advance.
best regards

Thank you very much for your prompt reply. Yes, I have tried CD68 wiith a positive and negative control section. As positive and negative control bovine lymph node samples were used. But the problem is that whenever I used the combination of isopropanol and Hcl for fixation and hot (60 degree celcius) PBS (+TWEEN) or methanol or xyline to remove the lipid droplets, I did not find any colour reaction in the positive control section. Here I would like to mention that I have to follow the same treatment which I used in case of viceral fat (to get rid of fat) also for positive and negative control section. Therefore I have to optimize it by using solution(s) which can remove fat without destroying CD68 epitopes. If you have any suggestion it will be helpful for me.
Best regards

I don't really know why you are losing antigenicity. I've come across some pretty harsh antigen retrieval protocols with CD68 IHC, on paraffin and frozen sections. This ref link shows IHC in adipose tissue on both paraffin and frozen sections. The frozen sections were fixed in acetone. There's a picture of the stains also.

Also, here's an interesting protocol that starts with frozen sections, fixes in acetone, then incubates in paraformaldehyde: For immunohistochemistry, frozen synovial tissues embedded in OCT compound were cut into 5-µm sections, fixed in cold acetone for 10 minutes, dried for 30 minutes, and soaked in 1% paraformaldehyde solution for 3 minutes. Endogenous peroxidase was blocked with 0.3% H2O2 in 0.1% sodium azide. Nonspecific binding was blocked with normal rabbit serum or normal goat serum. The following Abs were used for immunohistochemistry: goat polyclonal anti-TSP2 (1:300, Santa Cruz Biotechnology); mouse monoclonal anti-CD146 (1:300, P1H12; Chemicon, Temecula, CA); mouse monoclonal anti-CD3 (1:300, UCHT1; DAKO, Carpinteria, CA); and mouse monoclonal anti-CD68 (1:100, PG-M1; DAKO). Sections were incubated with primary Abs for 1 hour at room temperature. Biotinylated rabbit anti-goat IgG (Vector Laboratories) and goat anti-mouse IgG (1:200, DAKO) were used as secondary Abs. Sections were developed with Vectastain Elite ABC peroxidase reagents and 3,3'-diaminobenzidine tetrahydrochloride (Sigma-Aldrich). For double labeling, the sections were incubated with a second primary Ab and developed using Vectastain ABC alkaline-phosphatase reagents and Vector Red or Vector Blue (Vector Laboratories). For all staining experiments, control stains with isotype-matched Ab were included. Here's the link link

Best of luck.

Thank you very much for your prompt reply. Yes, I have tried CD68 wiith a positive and negative control section. As positive and negative control bovine lymph node samples were used. But the problem is that whenever I used the combination of isopropanol and Hcl for fixation and hot (60 degree celcius) PBS (+TWEEN) or methanol or xyline to remove the lipid droplets, I did not find any colour reaction in the positive control section. Here I would like to mention that I have to follow the same treatment which I used in case of viceral fat (to get rid of fat) also for positive and negative control section. Therefore I have to optimize it by using solution(s) which can remove fat without destroying CD68 epitopes. If you have any suggestion it will be helpful for me.
Best regards

Hi
Good day. I have tried with methanol or ice cold acetone or ice cold isopropanol several times for fixation of visceral fat for CD68 IHC. Then after 1 hr drying, I tried by treating the sections sometimes with warm PBS (0.05%, pH 7.2) (+TWEEN) (38 degree celcius) or sometimes with normal citrate buffer solution (0.01%, pH 6) for 5 minutes but it did not work. I mean fat remain inside the fat vacuole still, so that antibodies does not work properly. Another problem is that hot PBS (60 degree celcius) can remove the fat but I did not get any colour reaction, it destroy the CD68 epitope may be (I am not sure). Have you any suggestion how can I get rid of fat for visceral adipose tissue (frozen section) without destroying the CD68 epitope for detection of macrophage into it.
Thnaks in advance.

Have you ever gotten the antibody to work on non-fatty sections of any kind? For example, "Bone marrow macrophages and mast cells, liver Kupffer's cells, lung macrophages, lymph node and tonsil germinal center macrophages" (from link). If not, you might want to work out the conditions on one of these tissues/cells first. That way, you can tell which protocols result in loss of signal.

Hi
Good day. I have tried with methanol or ice cold acetone or ice cold isopropanol several times for fixation of visceral fat for CD68 IHC. Then after 1 hr drying, I tried by treating the sections sometimes with warm PBS (0.05%, pH 7.2) (+TWEEN) (38 degree celcius) or sometimes with normal citrate buffer solution (0.01%, pH 6) for 5 minutes but it did not work. I mean fat remain inside the fat vacuole still, so that antibodies does not work properly. Another problem is that hot PBS (60 degree celcius) can remove the fat but I did not get any colour reaction, it destroy the CD68 epitope may be (I am not sure). Have you any suggestion how can I get rid of fat for visceral adipose tissue (frozen section) without destroying the CD68 epitope for detection of macrophage into it.

Thnaks in advance.

Thank you very much indeed

You're welcome.

Thank you very much indeed

Hi
I am working with CD68 immunohistochemistry on adipose tissue to detect macrophage in it. I fixed adipose tissue frozen sections in ice cold isopropanol for ten minutes, then after 2 hrs drying I used hot PBS with TWEEN to remove the fat from the vacuole of fat cell, then I followed the actual protocol for CD68 immunohistochemistry, would you please suggest me whether this hot PBS destroy the antogenicity of adipose tissue or not. Because I did not find any colour reaction in positive control. And I would like to get the standard protocol for CD68 IHC on adipose tisse macrophages. Any suggestion will be highly appreciated. Thanks in advance.
Sorry for any inconvenience.
My Email Address: hasinavahbau@gmail.com

I don't have specific information on whether your protocol could damage the CD68 epitope but I have found several protocols that don't use a heated buffer treatment. Here's one:
Immunohistochemical detection of macrophages was performed on frozen 8-eeCthick human adipose tissue cryostat sections and fixed in 4% paraformaldehyde for 5 min, followed by methanol for 5 min. The sections were blocked for endogenous peroxidase activity by incubation in 0.3% H2O2 in PBS overnight at 4°C. For CD68 immunostaining, mouse anti-human CD68 monoclonal antibody (clone EBM-11; Dako, Milan, Italy) was used at a dilution of 1:150 in blocking solution (ImmPRESS Reagent Anti mouse Ig; Vector Laboratories, Burlingame, CA) overnight at 4°C, followed by peroxidase-coupled anti-mouse IgG (ImmPRESS Reagent; Vector labs, Burlingame, CA) for 1 h at room temperature. (Diabetes August 2005 vol. 54 no. 8 2305-2313).

Another:
Adipose tissue, muscle, and liver samples were fixed for 12–16 hours at room temperature in zinc-formalin fixative (Anatech Ltd., Battle Creek, Michigan, USA) and embedded in paraffin. Five-micron sections cut at 50-µm intervals were mounted on charged glass slides, deparaffinized in xylene, and stained for expression of F4/80 as described by Cecchini et al. (43) with an anti-F4/80 monoclonal antibody provided by E. Richard Stanley (Albert Einstein College of Medicine), or for expression of CD68 with the commercially available monoclonal antibody PG-M1 according to the manufacturer’s instructions (Dako CytoMation, Carpinteria, California, USA). For each individual mouse adipose depot, four different high-power fields from each of four different sections were analyzed. The total number of nuclei and the number of nuclei of F4/80-expressing cells were counted for each field. The fraction of F4/80-expressing cells for each sample was calculated as the sum of the number of nuclei of F4/80-expressing cells divided by the total number of nuclei in sections of a sample. The same procedure was used to measure the fraction of CD68-expressing cells in human tissues. Adipocyte cross-sectional area was determined for each adipocyte in each field analyzed using image analysis software (SPOT version 3.3; Diagnostic Instruments Inc., Sterling Heights, Michigan, USA). Average adipocyte cross-sectional area was calculated for each animal using Microsoft Excel (Microsoft Corp., Redmond, Washington, USA). (J. Clin. Invest. 112(12): 1796-1808 (2003))

It could be that the solvent treatments (methanol or xylene) after fixation help to get rid of the fat.
Might be worth a try.

Hi
I am working with CD68 immunohistochemistry on adipose tissue to detect macrophage in it. I fixed adipose tissue frozen sections in ice cold isopropanol for ten minutes, then after 2 hrs drying I used hot PBS with TWEEN to remove the fat from the vacuole of fat cell, then I followed the actual protocol for CD68 immunohistochemistry, would you please suggest me whether this hot PBS destroy the antogenicity of adipose tissue or not. Because I did not find any colour reaction in positive control. And I would like to get the standard protocol for CD68 IHC on adipose tisse macrophages. Any suggestion will be highly appreciated. Thanks in advance.
Sorry for any inconvenience.
My Email Address: hasinavahbau@gmail.com

Have you tried a mac-1 antibody? I've used mac-1 abs to stain mouse bone marrow for macrophages and have had no problem with background staining (I believe it was from BD Pharmingen).

Also, I would definitely recommend blocking with normal serum from the host species of the secondary antibody.

I was thinking about you problem some more, when we do immunofluorescense staining of fixed cells (not tissues), we usually use normal serum (in 2% FCS) as a block instead of BSA.

For example, if your primary antibody is a rabbit antibody and your secondary is a Donkey anti-Rabbit, we block with normal donkey serum. So anything that will bind donkey antibodies will already be blocked before you add the labeled secondary.

I am not sure if they do this for fixed tissue staining, but I would figure you have block with something more specific than just BSA.

Hope this helps

It appears they sell anti-mouse CD68 antibodies, here is a list of some sources:

link

I haven't done any immunohistochemistry, so I don't know if I can really help you out much. Do you have to block Fc receptors when you stain fix sections? Could this explain why the secondary alone gives you staining?

If you want to stain macrophages, can you use CD11b or is it not specific enough?

just some thoughts.