Re: [Histonet] mouse femour section
Hi Mani, Chondrocytes? Osteoclasts? Adipocytes? Send a pic and I will try to determine. Sent from my Verizon Wireless BlackBerry -Original Message- From: Rene J Buesa rjbu...@yahoo.com Sender: histonet-boun...@lists.utsouthwestern.edu Date: Thu, 26 May 2011 12:58:53 To: mani kandancoralm...@yahoo.co.in; histonet@lists.utsouthwestern.eduhistonet@lists.utsouthwestern.edu Reply-To: Rene J Buesa rjbu...@yahoo.com Subject: Re: [Histonet] mouse femour section Why don't you look in a histology atlas and try to find out. For your description they can be several things. René J. From: mani kandan coralm...@yahoo.co.in To: histonet@lists.utsouthwestern.edu Sent: Thursday, May 26, 2011 12:30 PM Subject: [Histonet] mouse femour section hai, i am working with mouse femours, i found some cells in the section looks like a ring shaped cells i want to know what r they,can u help me in this regard. M.Manikandan, Researcher, Stemcell unit, King Saud university, Riyadh,KSA +966552012697 ___ 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 mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Tumors tumors everywhere
Did you check to ensure the secondary in the kit is anti-rabbit IgG, with minimal cross to mouse? Ask Biocare directly. What does the background look like? What does your isotype look like? How about your secondary only control? Sent from my Verizon Wireless BlackBerry -Original Message- From: sgoe...@mirnarx.com Sender: histonet-boun...@lists.utsouthwestern.edu Date: Thu, 28 Apr 2011 15:16:16 To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Tumors tumors everywhere So I am staining tumors that were implanted as cells subdermally into mice. The cells are human. I am trying to do Caspase staining on these tumors. The primary is an anti-human rabbit polyclonal, and I am using a polymer (Biocare Mach3) in lieu of the secondary antibody. The background is through the roof!! Could the reason be that the tumor was grown in a mouse and is having cross reactivity somehow? What species antibody should I be using instead? All my mouse monoclonal antibodies work perfect on the tissue, it's this stupid rabbit polyclonal!! I am blocking endogenous enzymes (peroxidase etc., DAKO), avidin and biotin (just to see if that would help...it didn't), and protein block (it's literally an hour worth of blocking!!), developing with DAB (Dako) and hematoxylin counterstain. I am so confused as how to get this to work! Also, it isn't just this particular antibody it is any rabbit polyclonal I have tried. Could it be the polymer? It is the one that Biocare suggested? HELP!! Thanks in advance =) Sarah Goebel, BA, HT(ASCP) Histotechnologist Mirna Therapeutics 2150 Woodward Street Suite 100 Austin, Texas 78744 (512)901-0900 ext. 6912 ___ 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] human on human IHC
Along those same lines I have conjugated primaries to biotin and it works
beautifully for human on human IHC. Another alternative if extra amplification
is needed is to conjugating to FITC and then using a rabbit-anti-FITC followed
by an anti-rabbit polymer.
Just be aware with human on human controls are imperative as you may get
binding to human Fc receptors.
Andrea Hooper
Sent from my Verizon Wireless BlackBerry
-Original Message-
From: sgoe...@xbiotech.com
Sender: histonet-boun...@lists.utsouthwestern.edu
Date: Thu, 15 Jul 2010 09:27:19
To: Kim Merriamkmerriam2...@yahoo.com
Cc: Histonethistonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] human on human IHC
I am doing alot with this right now. The best thing I havefound to
do is to conjugate your primary antibody with HRP, then just use
the direct method (no secondary), and it turns out beautifully!!
You can buy thekit to do this from Fisher. I can get the exact
order number if you need it. Good Luck!!
Sarah Goebel, B.A., HT (ASCP)
Histotechnician
XBiotech USA Inc.
8201 East Riverside Dr. Bldg 4 Suite 100
Austin, Texas 78744
(512)386-5107
Original Message
Subject: [Histonet] human on human IHC
From: Kim Merriam [1]kmerriam2...@yahoo.com
Date: Thu, July 15, 2010 8:00 am
To: Histonet [2]histo...@lists.utsouthwestern.edu
Hi,
Does anyone know of a human-on-human IHC kit similar to the
mouse-on-mouse kits
that are available?
Thanks,
Kim
Kim Merriam, MA, HT(ASCP)QIHC
Cambridge, MA
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Re: [Histonet] air dry or heat dry
I recommend to just take out the slides you need one at a time. If you are concerned about icy slides take them out rapidly without removing the box from the freezer (ie: do it on the shelf in the -80 deg). Also add some dessicant to the slide box. If its a major concern, use smaller slide boxes, or cut less slides, or individually heat seal each slide or small set of slides into their own plastic envelopes. I cut the OCT slides. Dry for about 4h then put at -80 deg C. After freezing I do not allow the slides to warm at all. They go directly from the slide box in freezer into the PFA (or PBS depending). Great morphology. I do not recommend you heat your sections once, much less the whole box repeatedly. This seems like a bad idea over time for sure. Especially if you are trying to repeat experiments and conditions. Why do you acetone fix when your tissues are already PFA fixed? This seems unnecessary. If I had to choose from your selection the best technique, I say the air dry group in the hood for 30 min is best. Andrea Sent from my Verizon Wireless BlackBerry -Original Message- From: Jeffrey Thompson jefthomp...@salud.unm.edu Sender: histonet-boun...@lists.utsouthwestern.edu Date: Fri, 09 Jul 2010 15:21:47 To: histonet@lists.utsouthwestern.edu Subject: [Histonet] air dry or heat dry Hello, We have an ongoing debate in our lab regarding the relative virtue of heat drying slides vs RT air drying them. Tissues: 4% paraformaldehyde perfused rat brains frozen in OCT blocks Sections: 10 microns on a cryostat (at approx -25 C) Slides: Superfrost Plus The slides are stored in slide boxes at -80 C until staining. When staining the heat dry faction (wanting to avoid icy slides) put their slide boxes straight out of the freezeer into a 50 C oven for 30 minutes before taking out slides to stain. then the box goes back to the freezer until the next round of staining, The air dry group feels that cooking the antigens repeatedly at 50 C is problematic so they take the frosty slides out their slide boxes and return them ASAP to the freezer. The slides to be stained are air dried in the fume hood for about 30 min. A third, middle of the road, person takes her slides out of the cold boxes and then puts the slides in a 60 C oven for 30 minutes. For all three groups then, the slides are given a PAP pen border to prepare for IHC and when the pen solution is dry, 10 minutes in acetone and the remainder of the staining procedure. So my question is: Who is using the best technique? Another hotly debated topic is wether it is advisable to put a drop of PBS on the slide before 'sticking' the section to prevent folds in the sections.. Any opinions are appreciated. Thanks, Jeff ___ 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] VEGFR2 + VEGFR3
What is everyone's current favorite anti-VEGFR2 and anti-VEGFR3 antibody for staining human tissue? As usual I am in the market for some new reagents. Much appreciated, Andrea Sent from my Verizon Wireless BlackBerry ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] IHC
Overnight is surely fine but make sure at 4 deg C for two reasons. First you want to minimize any bacterial growth. Second you want to ensure it doesn't evaporate significantly which would change your antibody concentration. For that same reason make sure you humidify the chamber amply as well as don't skimp on the volume applied to the slide - as even humidified and at 4 deg C eventually everything evaporates (say if you left it for days). If you routinely do this ab for 1h RT and are now switching to overnight be aware you may (or may not) get more staining or nonspecific binding. The titration of ab could be a little different when optimiZed for overnight. That being said, I say go for it. Sent from my Verizon Wireless BlackBerry -Original Message- From: sgoe...@xbiotech.com Date: Fri, 14 May 2010 14:37:19 To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC Can you leave your primary antibody on slides overnight (or the w eekend) or will this screw things up? I don't want to be here anothercouple of hours for secondary, etc. on a Friday!! Sarah Goebel, B.A., HT (ASCP) iHistotechnician XBiotech USA Inc. 8201 East Riverside Dr. Bldg 4 Suite 100 Austin, Texas 78744 (512)386-5107 ___ 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 Doublestaining
I completely agree with all Adam has said. The essential key is to make sure your secondaries are highly cross adsorbed to each other and including mouse since you are staining mouse stroma. Also run each as singles alongside your double - that will give you great confidence in what you are seeing is real. Should be a piece of cake ;) Sent from my Verizon Wireless BlackBerry -Original Message- From: Adam . anonwu...@gmail.com Date: Thu, 13 May 2010 13:43:57 To: Igor Deynekoigor.deyn...@gmail.com Cc: histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] IF Doublestaining In my experience, most fluorophores are quite stable to many washes. Here is what I would try at first: 1) Stain with rat anti-mouse and rabbit anti-human overnight at 4C in a humidified chamber. Dilute each antibody in a single volume of your favorite staining buffer (PBS, TBS, TBS-T). For example, if your rat antibody needs to be diluted in 1:100 and your rabbit needs to be diluted 1:200, take an aliquot of 200 uL of buffer and add 2 uL of the rat primary and 1 uL of the rabbit. Add the appropriate volume (usually 50-200 uL) of that mixture to each slide. 2) Wash the next day, and add your secondaries again to a single mixture for 1 hour at room temperature. As long as your secondaries have been highly cross adsorbed to many species, you shouldn't have a problem. 3) Wash, counterstain, mount, enjoy. I really don't think you need to do sequential staining for this. I've heard anecdotal reports of two primaries or two secondaries forming immune complexes when mixed together, but I've never really had that problem. Good luck, Adam On Thu, May 13, 2010 at 1:00 PM, Igor Deyneko igor.deyn...@gmail.comwrote: Hello Everyone! I'm planning to try some IF co-staining with 2 antibodies, one is a rat-anti-mouse and the other one is rabbit anti-human on a xenograft, each has an appropriate secondary, donkey anti rat and donkey anti-rabbit, conjugated to 488 and 593. Can someone advise the best way to perform such a procedure, I'm afraid the rules of sequential staining might not work due to fluorophore instability with washes. if anyone has performed such type of stain, i would appreciate any tips. Thank you. Igor Deyneko Infinity Pharmaceuticals Cambridge, MA 02139 ___ 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 mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
