[Histonet] Cryotomy help
I assume that the fresh tissue was fixed in methanol? If so, it is irreversibly coagulant- fixed ( like boiling an egg white...no going back) Place the specimen in dist water until it melts to RT x3 changes of dw 1hr each at RT on a gentle rocker to get rid of methanol ( antifreeze, hence inability to freeze as you have replaced water) Place in OCT at RT on a rocker for 30 mins Orientate and snap-freeze Or, if your mass spec doesn't like OCTfreeze without OCT ? NB: Most important to freeze quickly to avoid ice-crystal formation Proper snap-freezing is used to make the water go from liquid to vitreous ( glass-like) state, avoiding the ice-crystal state ( which will occur with SLOW freezingthus giving you horrible ice-crystal artefact) Good luck! Carl Hobbs FIBMS Histology and Imaging Manager Wolfson SPaRC Guys Campus, London Bridge Kings College London London SE1 1UL 020 7848 6810 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] What is the best xylene substitute for histology?
As usual, great insights/advice from JK I always learn from you, Sir You are THE person that I "listen" to ( you may have stood on the shoulders of others?? Do tell) I'm always looking for xylene substitutes.haven't found any as efficientsigh I run the gamut with my Safety people regularly but.they don't understand Then they just "disappear" each year Not good, I know I run a Leica "dipN dunk" processor ( Leica TP1020) Been using it for 20 yrs without problems...phew! However.many diagnostic labs use ( far more expensive) substitutes effectively Homing in to this thread/topicavidly Carl Hobbs FIBMS Histology and Imaging Manager Wolfson SPaRC Guys Campus, London Bridge Kings College London London SE1 1UL 020 7848 6810 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Alcohol
For 50 yrs I have used Industrial methylated spirit ( 74OP IMS) It is cheaper than ethyl alcoholdoes the same job as it is ethyl alcohol containing a part of methyl alcohol No duty charged Until recently I would buy 25 L drum Carl Hobbs FIBMS Histology and Imaging Manager Wolfson SPaRC Guys Campus, London Bridge Kings College London London SE1 1UL 020 7848 6810 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] [EXT] Reagent alcohols and tissue
I ask Researchers to give me cassettes in 90% alcohol as I start my "dipN dunk" processor ( Leica TP 1020) at 90% They fix, rinse in tap water, dist water, x2 70% alcohol, then bring to me in 90% alcohol This way, less likely to contaminate my 90% alc with their inadequate alcohol rinses. Been doin it for 30 yrsOK so far Carl Carl Hobbs FIBMS Histology and Imaging Manager Wolfson SPaRC Guys Campus, London Bridge Kings College London London SE1 1UL 020 7848 6810 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] tissue cassettes
Why would anyone use a different wax for infiltrating and embedding? Yeskeep them specimens molten until embedded Thanks for your input, Paula Time flies like an arrow Fruit flies like a banana BonBon-illy Carl ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] tissue cassettes
I'm interested but don't understand the variation and it's + effect I take my cassettes out of the processor and immediately place into the molten wax bath of the embedder ( if I'm embedding immediately; if not I let the cassettes/tissues therein go cold until a later embedding) When embedding immediately, after 30 mins, I remove tissue from cassette into a heated metal mold filled with molten wax All done quickly, to keep all wax molten If my people take too long, the wax around the tissue sets so when the tissue is placed into the molten wax, in mold, they sometimes get that effect of set wax-interface artefactthe section immediately pulls apart from the surrounding wax, on the waterbath This can cause the section to fold Do enlighten me Thanks Carl Never too old to learn! Carl Hobbs FIBMS Histology and Imaging Manager Wolfson SPaRC Guys Campus, London Bridge Kings College London London SE1 1UL 020 7848 6810 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] DAB freezing (Alonso Mart?nez Canabal)
I do manual IHC-DAB regularly DAB shouldn't "go off" after freezing unless you have somehow oxidised it during mixing/aliquoting. It would then show as a dark brown aliquot I state this because I still make up my own DAB from dry powder ( have been doing for > 20yrs) SIgma D5637 I dissolve, aliquot and freeze, as you do. An aliquot may be stored at -20C for 2yrs before I defrost/use it If I run out of 50microL aliquots I will defrost a 1ml aliquot, re-aliquot into 50 microL ( when I am doing IHC on a few slides, I make up a small working DAB vol.) Perhaps your H2O2 ( substrate) stock has "gone off"? That will give you a weaker/negative DAB result NB: left-over working DAB solution can be stored at 4C for at least 7 days and still be perfectly usable ( NOT re-usable) If I have only 5-10 slides I will do the DAB step in the humidity chamber, applying DAB as I would antibody (only into the hydrophobic pen-ringed section) Good luck and...please let us know the answer when you solve your problem Carl Hobbs FIBMS Histology and Imaging Manager Wolfson SPaRC Guys Campus, London Bridge Kings College London London SE1 1UL 020 7848 6810 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] IF staining questions (Charles Riley)
The primary antibody is the most expensive reagent If you use the primary ab unconjugated and visualise using a conjugated secondary you will significantly increase the dilution factor of the primary ab thus your primary ab will last much longer ( saving you money..and, maybe less background noise Same principle applies to using antigen retrieval( AR) v no ag AR: eg: I can use a particular ab to detect "endogenous" GFP ( actually tagged to a protein) in Pwax sections without using AR ( actually HIER) at a dilution factor of 1/750 If I subject the section to HIER, I can use the same primary ab at a dilution factor of at least 1/2K Same applies to 90C HIER v M/W HIER: former requires 30 mins or more, latter requires ½ that time and, is more effective overall ( stronger signal/greater diln factor) Imho Good luck! Carl Carl Hobbs FIBMS Histology and Imaging Manager Wolfson SPaRC Guys Campus, London Bridge Kings College London London SE1 1UL 020 7848 6810 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] maximum thickness brain in slide
Hi Alonso Firstly, what antibody( ies) are you wanting to use on your Pwax sections? If you are looking just to identify proteins, 8 microns is sufficient I mount my brain sections ( human, ms, rat, axolotl, worm, fruitfly, pig...etc) then airdry them O/N in a working fume hood Then, before dewaxing, I put them into a 60C oven for 1hr. Imho, free-floating sections are useful only if you wish to study projections over depth Use Superfrost Plus ( or equivilent) slides Good luck! Carl Carl Hobbs FIBMS Histology and Imaging Manager Wolfson CARD Guys Campus, London Bridge Kings College London London SE1 1UL 020 7848 6810 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
