http://www.protocol-online.org/forums Wed, 08 Feb 2012 08:03:45 +0000 3600 http://www.protocol-online.org/forums/topic/24329-isolation-of-neutrophil-from-mice-blood/
Thanks]]> Wed, 08 Feb 2012 08:03:45 +0000 http://www.protocol-online.org/forums/topic/24329-isolation-of-neutrophil-from-mice-blood/ http://www.protocol-online.org/forums/topic/24226-blood-to-serum-ratio-in-rats/
Will]]> Tue, 31 Jan 2012 13:10:46 +0000 http://www.protocol-online.org/forums/topic/24226-blood-to-serum-ratio-in-rats/ http://www.protocol-online.org/forums/topic/24034-preparation-of-tibia-or-femur-for-microct/
Actually I'm looking for convenient protocols for preparation of tibia and femur of mice. The bone will be analyzed by micro CT for determination of the trabecular bone volume and by pq-CT for bone mineral density.
I suggest that these protocols are quite simple, however I'm uncertain whether I should use protocols utilizing PFA for fixation, just Ethanol or even KOH to get rid of connective and muscle tissues. In addition, I’m not sure about storing the bone before CT at 4°C or -20°C ?

Thanks
Alex]]> Mon, 16 Jan 2012 14:45:49 +0000 http://www.protocol-online.org/forums/topic/24034-preparation-of-tibia-or-femur-for-microct/ http://www.protocol-online.org/forums/topic/23882-littermate-control-mice/
colud anyone explain me what "Littermate control mice" are?

I've never worked with mice...

Thanks in advance!!]]> Mon, 02 Jan 2012 22:16:37 +0000 http://www.protocol-online.org/forums/topic/23882-littermate-control-mice/ http://www.protocol-online.org/forums/topic/23799-can-i-use-female-rats-instead-of-male-ones/ I want to investigate the effect of a herbal extract on rats. I have ssen in papers that male wistar rats have been used for similar experiments. I have lots of female rats. can I use them or should I use male rats. Besides, why in most of the articles male rats have been used and whats the difference?
Thanks in advance.]]> Sat, 17 Dec 2011 05:47:26 +0000 http://www.protocol-online.org/forums/topic/23799-can-i-use-female-rats-instead-of-male-ones/ http://www.protocol-online.org/forums/topic/23638-mouse-breeding-double-kos/
I'm trying to create double KOs, mice that are knockouts for both gene A and gene B. I started by breeding single knockouts, so I crossed a knockout for gene A (A -/-) with a knockout for gene B (B -/-). Therefore the first generation of pups was hetero for each gene. Then, I crossed this first generation of pups with itself, trying to get double KOs.

The interesting thing is what happened when I genotyped the second generation of offspring. The cross was hetero (A+/- w/ B+/-) so I expect 25% homozygous +/+, 50% hetero +/-, and 25% homozygous -/-. BUT what I get from 14 offspring is:

ALL mice homozygous -/- for gene B
7 mice homozygous +/+ for gene A
and 7 mice either heterozygous +/- or homozygous -/- for gene A (can't tell the difference between +/- and -/- with the PCR protocol)

So does anyone know why ALL the second generation mice are genotyped to be -/- for gene B but only potentially 7 out of 14 mice are genotyped to be -/- for gene A? Why the difference in frequency between gene A and gene B?

Thanks!

Janey]]> Thu, 01 Dec 2011 16:49:03 +0000 http://www.protocol-online.org/forums/topic/23638-mouse-breeding-double-kos/ http://www.protocol-online.org/forums/topic/23360-hoyers-mounting-media/
Thanks]]> Tue, 08 Nov 2011 17:40:51 +0000 http://www.protocol-online.org/forums/topic/23360-hoyers-mounting-media/ http://www.protocol-online.org/forums/topic/23357-how-to-measure-radioactive-cholesterol-3h-in-c-eleganas/
a referee has asked to measure radioactive cholesterol uptake in C. elegans, does any have any idea of how to do this? Perkin Elmer sells cholesterol with tritium and 14C, I believe is better to use 3H , any ideas?? witch scintillation liquid is best with C. elegans or how many worms should I use per vial? do you think that ONE single worm will be measurable?
Thanks a lot]]> Mon, 07 Nov 2011 19:25:47 +0000 http://www.protocol-online.org/forums/topic/23357-how-to-measure-radioactive-cholesterol-3h-in-c-eleganas/ http://www.protocol-online.org/forums/topic/23320-organ-perfusion-spleen-perfusion/
I'm experiencing problems in the perfusion of mouse spleen. I'd like to separate resident splenocytes from circulating leukocytes in the spleen. I tried to use standard techniques, like perfusion with PBS from the heart (under terminal anaesthesia) or perfusion from the aorta. Big organs like lungs and liver are cleared, but the spleen remains full of blood cells (probably due to the peculiar vascular anatomy).

Has anyone noticed spleen whitening during organ perfusion with other techniques???

thanks]]> Thu, 03 Nov 2011 19:14:07 +0000 http://www.protocol-online.org/forums/topic/23320-organ-perfusion-spleen-perfusion/ http://www.protocol-online.org/forums/topic/23230-why-is-my-cre-pcr-data-does-not-match-the-immunofluorescent-labeling/
Have you experienced this: you genotyped a mouse (ear clip) and found it to be Cre-positive; then you take the tissue and do immunostaining but there's no labeling with the Cre antibody!

I have a positive control making sure the Cre antibody and my method works.

Any thoughts?

Thanks!]]> Wed, 26 Oct 2011 23:13:02 +0000 http://www.protocol-online.org/forums/topic/23230-why-is-my-cre-pcr-data-does-not-match-the-immunofluorescent-labeling/ http://www.protocol-online.org/forums/topic/22621-invertebrate-injectionsmicroinjections-nitrogen-vs-compressed-air/
Thanks in advance.]]> Sat, 27 Aug 2011 22:33:01 +0000 http://www.protocol-online.org/forums/topic/22621-invertebrate-injectionsmicroinjections-nitrogen-vs-compressed-air/ http://www.protocol-online.org/forums/topic/22272-isolation-of-bursa-of-fabricius-from-chicken/
Attempts to isolate primary cells from the bursa of Fabricius failed due to high event of contamination .
Any suggestion or creative ways to avoid the contamination will be very much appreciated.
Thank you ]]> Tue, 26 Jul 2011 02:22:02 +0000 http://www.protocol-online.org/forums/topic/22272-isolation-of-bursa-of-fabricius-from-chicken/ http://www.protocol-online.org/forums/topic/22167-thioglycollate-for-pmn-collections/
I need help with making thioglycollate for mice injections for collecting PMNs. I know 4% of it is required for injections. The label of the powder that we have says to dissolve 29g in 1L. Shall I have to dissolve 40g/L to make 4%?

Any help would be appreciated.

Thanks..]]> Tue, 19 Jul 2011 03:18:34 +0000 http://www.protocol-online.org/forums/topic/22167-thioglycollate-for-pmn-collections/ http://www.protocol-online.org/forums/topic/21662-nematocysts-isolation/
does anyone has a good way of nematocysts isolation from cnidaria?]]> Wed, 15 Jun 2011 07:49:01 +0000 http://www.protocol-online.org/forums/topic/21662-nematocysts-isolation/ http://www.protocol-online.org/forums/topic/21585-rehydration-procedures-for-preserved-fish/
In my lab I am dissecting various species of fish and weighing their individual organs. The fish I am working with have been initially fixed in formalin and then transferred to 70% ethanol. However, the ethanol quickly evaporates during the dissection and weighing stages, preventing me from obtaining accurate measurements. I was thinking of gradually transferring the fish from 70% ethanol --> 50% ethanol --> 30% ethanol --> 15% ethanol --> 0% ethanol (all in 0.1M phosphate buffer, too), but I'm not sure how long I should keep the fish in each solution before transferring it to the next. I want to give them enough time to rehydrate. Would 24 hours suffice? These fish all weigh between 10 and 20 grams, so they're not very large

Thanks!]]> Fri, 10 Jun 2011 18:02:42 +0000 http://www.protocol-online.org/forums/topic/21585-rehydration-procedures-for-preserved-fish/ http://www.protocol-online.org/forums/topic/21578-acidic-water-preparation-for-mice/ One more thing, i also heard that acidic water cannot be keep in glass bottle, i just want to know the reason....

thank..]]> Fri, 10 Jun 2011 11:31:17 +0000 http://www.protocol-online.org/forums/topic/21578-acidic-water-preparation-for-mice/ http://www.protocol-online.org/forums/topic/21575-tissues-used-to-genotyping-animals/
What tissues does everyone here take for genotyping? For mice I've read the common one is either tail or toe snip. I'm about to do some behavioral work and am hesitant to essentially mutilate my mice.

Moreover, has anyone had success with using stool samples for genotyping purposes? There is an interesting paper on it, but I am unsure of how successful it would be for me: http://www.ncbi.nlm.nih.gov/pubmed/10580111]]> Fri, 10 Jun 2011 07:16:48 +0000 http://www.protocol-online.org/forums/topic/21575-tissues-used-to-genotyping-animals/ http://www.protocol-online.org/forums/topic/21572-drosophila-germline/
It is my understanding that germline and somatic expression of transgenic constructs, etc. is quite different. I have two questions: First, what makes the two so different? And second: when I am looking up stocks, etc. (not necessarily drivers, but any transgenic line) how do I know if it expresses well in the germline? Any help would be appreciated!!]]> Fri, 10 Jun 2011 02:19:29 +0000 http://www.protocol-online.org/forums/topic/21572-drosophila-germline/ http://www.protocol-online.org/forums/topic/21358-mouse-recoder-software/
my current method is writing them down in the notebook.

So, I come to ask where somebody known some awesome software to record the breeding mice ?


Thks.]]> Sun, 29 May 2011 12:17:05 +0000 http://www.protocol-online.org/forums/topic/21358-mouse-recoder-software/ http://www.protocol-online.org/forums/topic/21170-mass-antibody-producers/
I am looking for large amounts of antibody (about 50 mg).
My experiences consist in depleting mice for CD4/CD8.

Could you indicate me a list of providers which might have this type of product ?

Thank you in advance.]]> Thu, 19 May 2011 06:58:43 +0000 http://www.protocol-online.org/forums/topic/21170-mass-antibody-producers/