Re: [Histonet] IF staining on peritoneal macrophages
Dear Mauricio, The two antibodies you chose were NOT cross adsorbed against mouse. This is critical for successful staining of mouse tissue with rat antibodies. These are the two alternate catalog #s of the ones I would recommend if FITC is your thing: 712-095-153 (whole IgG FITC conjugated) 712-096-153 (Fab'2 FITC conjugated) Although the important part to reduce background and increase sensitivity is the antibody itself, the choice of fluorophore can also play a huge role in your success. There are now many much better fluorophores than FITC and I suggest you play around with some of those. Firstly are you fixed on using the green channel (ie FITC) vs the red channel (ie rhoadmine)? I personally like CY3 (red) the best for brightness of the Jackson IR fluorophores. CY2 is also great. They also have a new series called DyLights which are reported to be great. I have played with them a little bit with some success and some failures. Ok, so assuming you want to stick with the green channel (I suggest you use CY2 then) these are the two simple choices for you (the second one is a F(ab')2 and one is a whole IgG - 99/100 the whole IgG will be fine for you and is what I use for staining hematopoietic tissue ...): 712-225-153 (whole IgG CY2 conjugated) 712-226-153 (Fab'2 CY2 conjugated) Another option for boosting signal is to use a biotinylated antibody and then use streptavidin conjugated to your fluorophore of choice. Then if you want to use the Alexa488s you can get streptavidin-Alexa488 from Invitrogen. 712-065-153 (whole IgG biotinylated) In my lab, I use both the CY2/CY3 conjugates as well as the biotin conjugates routinely. I rarely if ever use the F(ab')2 antibodies, it's normally overkill. Let me know if you need anything else, Andrea --- On Wed, 3/3/10, Mauricio Avigdor bitesizell...@gmail.com wrote: From: Mauricio Avigdor bitesizell...@gmail.com Subject: Re: [Histonet] IF staining on peritoneal macrophages To: Andrea T. Hooper andreahoo...@rocketmail.com, histonet@lists.utsouthwestern.edu Date: Wednesday, March 3, 2010, 8:18 PM Hi Andrea, Jackson lists a couple of FITC-conjugated donkey anti-rat secondaries: 712-095-150 - Whole Donkey Anti-Rat IgG (H+L). 712-096-150 - F(ab')2 fragment Donkey Anti-Rat IgG (H+L). Do you recognize which is the one you have ben using. I have been using Serotec's STAR80F antibody. They suggest using 1:10 Normal Mouse Serum in PBS to make the necessary dilutions. Is this something you do with the Jackson antibodies as well? I've never heard of this technique before. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] IF staining on peritoneal macrophages
Sorry for the second email, I didn't address all your questions ... The antibody I use from Serotec is MCA497, works very well and many people on Histonet have claimed the same over the years. I am not familiar with the secondary you use from Serotec. It says it's goat anti-rat IgG, mouse adsorbed. So it should be ok? But again FITC really isn't a great fluorophore and I tend not to use anything except Jackson IR reagents. I don't understand why they would suggest to use mouse serum, especially since it's mouse adsorbed? Unless you are trying to block Fc receptors - again, probably overkill. I have stained mouse hematopoietic tissue for years with rat antibodies and secondaries from Jackson and have never had this problem. I think this mouse serum step will just add background. Don't do it. Usually one is supposed to dilute their secondaries in the serum that the secondary is made in. Therefore in your case, I would use goat IgG. Block in your favorite blocking recipe ... For the antibodies I recommended to you, I use 10% donkey serum with 5% BSA (make sure it's gamma globulin free) in buffer with some detergent. Incubate primaries in 0.2X concentration of your block. If you want to share your protocol, maybe I can give you some further tips? Let me know if I can help further, Andrea --- On Wed, 3/3/10, Mauricio Avigdor bitesizell...@gmail.com wrote: From: Mauricio Avigdor bitesizell...@gmail.com Subject: Re: [Histonet] IF staining on peritoneal macrophages To: Andrea T. Hooper andreahoo...@rocketmail.com, histonet@lists.utsouthwestern.edu Date: Wednesday, March 3, 2010, 8:18 PM Hi Andrea, Jackson lists a couple of FITC-conjugated donkey anti-rat secondaries: 712-095-150 - Whole Donkey Anti-Rat IgG (H+L). 712-096-150 - F(ab')2 fragment Donkey Anti-Rat IgG (H+L). Do you recognize which is the one you have ben using. I have been using Serotec's STAR80F antibody. They suggest using 1:10 Normal Mouse Serum in PBS to make the necessary dilutions. Is this something you do with the Jackson antibodies as well? I've never heard of this technique before. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] IF staining on peritoneal macrophages
Rat anti-mouse F480 from serotec works hwell. It is crucial that you use a secondary against rat IgG that is highly cross adsorbed to many species, including mouse. I suggest whole IgG of donkey anti-rat IgG from Jackson Immunoresearch. Just make sure to pick the version adsorbed against mouse. I would give you the catalog number except I am not at my computer. Andrea T. Hooper ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] IF staining on peritoneal macrophages
Rat anti-mouse F480 from serotec works hwell. It is crucial that you use a secondary against rat IgG that is highly cross adsorbed to many species, including mouse. I suggest whole IgG of donkey anti-rat IgG from Jackson Immunoresearch. Just make sure to pick the version adsorbed against mouse. I would give you the catalog number except I am not at my computer. Andrea T. Hooper ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] IF staining on peritoneal macrophages
Hi Andrea, Jackson lists a couple of FITC-conjugated donkey anti-rat secondaries: 712-095-150http://www.jacksonimmuno.com/MERCHANT2/merchant.mv?Screen=PRODProduct_Code=712-095-150 - Whole Donkey Anti-Rat IgG (H+L). 712-096-150http://www.jacksonimmuno.com/MERCHANT2/merchant.mv?Screen=PRODProduct_Code=712-096-150 - F(ab')2 fragment Donkey Anti-Rat IgG (H+L). Do you recognize which is the one you have ben using. I have been using Serotec's STAR80F antibody. They suggest using 1:10 Normal Mouse Serum in PBS to make the necessary dilutions. Is this something you do with the Jackson antibodies as well? I've never heard of this technique before. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] IF staining on peritoneal macrophages
Hello Histoneters, I am very happy to say that my cytospins are finally working and that cells are staying on the slides throughout the procedure. Thank you very much to all who chimed in or helped with trial samples. I was almost ready to declare victory when I noticed one of my negative control slides looked a bit much like it has specific staining. The antibodies I have been using have not been giving the most even results. Does anyone have a recommendation for an anti-mouse F4/80 antibody? And a matching FITC-conjugated secondary antibody? Is anyone here doing IF staining of mouse peritoneal macrophages? ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] IF staining on peritoneal macrophages
After cytospinning I always dry my slides thoroughly before moving on to a IHC or IF step. I also use Superfrost Plus slides or silane coated slides to increase adherance. I would do that then try various fixatives. You can make a cell pellet as you suggest. Decide ahead of time if you want to fix before embedding (ie: is your antigen preserved in PFA). If so, fix in suspension then spin and resuspend in a small volume of 2% agarose (or Histogel). Then you can embed in OCT for frozen sectioning or wax for FFPE. - Andrea Hooper --- On Thu, 2/18/10, Mauricio Avigdor bitesizell...@gmail.com wrote: From: Mauricio Avigdor bitesizell...@gmail.com Subject: [Histonet] IF staining on peritoneal macrophages To: histonet@lists.utsouthwestern.edu Date: Thursday, February 18, 2010, 11:00 PM Greetings all, I am trying to do immunofluorescence on peritoneal macrophages. I am having a couple of issues that I was hoping one of you could help me resolve. Firstly, I am having uneven results with the Cytospin. Cells tend to get washed off the slides during rinses. Does anyone have tips on how to make cells stick a little better to Cytoslides? Secondly, I have not yet found a satisfactory method for fixation. In order to prevent loss of cells when dipping the slides into fixatives, I am having to air dry the slides before I can do anything to them. I tried the Shandon Collection Fluid (ethanol, isopropanol, carbowax) with great success, but it killed the fluorescence. I am leaning towards spinning the lavage fluid until I get a pellet and then resuspending in PBS with a little BSA added. I hope this makes cells stick well enough that I can put the slides in formalin or acetone. Any thoughts are appreciated! ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] IF staining on peritoneal macrophages
Thank you Adam and Jay for your replies. Cytoslides are the pre-marked slides for use with the Cytospin - http://www.thermo.com/com/cda/product/detail/0,1055,21035,00.html Adam, do you have a recommended concentration of BSA for this? Also, do you air dry the slides prior to fixation? Do you air dry afterwards? Thanks again for your help. Mauricio ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] IF staining on peritoneal macrophages
When I did it, I think I used our standard sort buffer, which is 0.2% BSA in PBS pH 7.4. I've also read that you can use anything with serum, typically 5-10%. The key seems to be prewetting the membranes with something with protein. I usually cytospin them and briefly (30 seconds) air dry them until there is no obvious liquid, before I added the fixative. I managed to get usable (but not ideal) cytospins from as few as 1000 cells. Let me know if it works, Adam On Fri, Feb 19, 2010 at 9:47 AM, Mauricio Avigdor bitesizell...@gmail.comwrote: Thank you Adam and Jay for your replies. Cytoslides are the pre-marked slides for use with the Cytospin - http://www.thermo.com/com/cda/product/detail/0,1055,21035,00.html Adam, do you have a recommended concentration of BSA for this? Also, do you air dry the slides prior to fixation? Do you air dry afterwards? Thanks again for your help. Mauricio ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] IF staining on peritoneal macrophages
You may want to try using the NuView Cyto prep technique from QC sciences it is a good alternative to a Cytospin ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] IF staining on peritoneal macrophages
Greetings all, I am trying to do immunofluorescence on peritoneal macrophages. I am having a couple of issues that I was hoping one of you could help me resolve. Firstly, I am having uneven results with the Cytospin. Cells tend to get washed off the slides during rinses. Does anyone have tips on how to make cells stick a little better to Cytoslides? Secondly, I have not yet found a satisfactory method for fixation. In order to prevent loss of cells when dipping the slides into fixatives, I am having to air dry the slides before I can do anything to them. I tried the Shandon Collection Fluid (ethanol, isopropanol, carbowax) with great success, but it killed the fluorescence. I am leaning towards spinning the lavage fluid until I get a pellet and then resuspending in PBS with a little BSA added. I hope this makes cells stick well enough that I can put the slides in formalin or acetone. Any thoughts are appreciated! ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] IF staining on peritoneal macrophages
The best way I know to get cells to stick is to first cytospin liquid with some protein (BSA or serum) onto the filters to pre-wet them. After that's done, cytospin your cells. I've gotten this to work with fairly rare FACS sorted cells. I've also found that the smaller volume the cells are in, the better your cytospin. Ideally is 100 - 200 uL. For FACS sorted cells, this means you want to sort into as small a volume as possible. For lavage cells, that may mean spinning them down first, although for FACS cells, the cells are fragile and don't like being centrifuged. I've gotten cells to stick to the slides by circling the cells with a PAP pen and then just putting fixative inside PAP while the slides are lying horizontally. Adam On Thu, Feb 18, 2010 at 5:00 PM, Mauricio Avigdor bitesizell...@gmail.comwrote: Greetings all, I am trying to do immunofluorescence on peritoneal macrophages. I am having a couple of issues that I was hoping one of you could help me resolve. Firstly, I am having uneven results with the Cytospin. Cells tend to get washed off the slides during rinses. Does anyone have tips on how to make cells stick a little better to Cytoslides? Secondly, I have not yet found a satisfactory method for fixation. In order to prevent loss of cells when dipping the slides into fixatives, I am having to air dry the slides before I can do anything to them. I tried the Shandon Collection Fluid (ethanol, isopropanol, carbowax) with great success, but it killed the fluorescence. I am leaning towards spinning the lavage fluid until I get a pellet and then resuspending in PBS with a little BSA added. I hope this makes cells stick well enough that I can put the slides in formalin or acetone. Any thoughts are appreciated! ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] IF staining on peritoneal macrophages
Mauricio, Sorry if this is too obvious, but are you using plus slides? I'm not sure what you mean by cytoslides. I always use plus slides with the Cytospin, and they stay on through HE, Pap, and DQ, although I'm not trying IF on them. Jay A. Lundgren M.S., HTL (ASCP) ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
