Nice one, Joelle.
Good points
I thank you for that sophisticated paper!
Yep, wax is not JUST wax.
It indeed has a crystalline, sophisticated structure.
Yes, cutting good sections depends upon temp/speed of cutting/angle of
knife/additives/effectiveness of processing.
May be more variables
I don't like ice-trays.
Well, I tried them and.they swelled me blocks!
Stick them blocks in the fridge, instead!
Sure, cooling them blocks is damn fine to get very thin kidney sections...
Diane!
The wonderful Chief Tech of Gt Ormond St kids Hospital Where are you???
You know all this
Hmmm
Nobody has yet mentioned the rationale behind the need to cool Pwax blocks.
Sure, it makes sectioning easier butnot always.
Never forget the huff!
Let's get back down to microtomy basics, so newbies have an appreciation that
sectioning is a mechanical process, as well as an
My bread and butter tissue preps are Pwax brainsoften serial sections but
not for stereology ( eg: mouse/chick/rat/zfish/monkey).
I routinely, like many others, cut serial sections varying between 10 to 200
sections ( more, if reqd)
They can be prone to chatter.
When this happens I
Assuming that you are fixing fresh-frozen tissue sections:
Tissue is autolysing.
Use Formalin for 10 mins or 1:1 acetone:alcohol for 10 mins.
Imho, acetone is not a fixative..it's a delipidizer.
5 mins in that mixture is too short a time for the alcohol to be an effective
coagulant fixer.
When you use smear, do you mean that the nuclei are blurring/ill-defined ,
with threads of DAPI-positivity coming away from the nuclei?
If so, it is due to underfixation.
Acetone is not a proper fixative.
In the time one uses is, it rapidly delipidises the tissue, rendering it better
preserved,
You could have a look here, in the image gallery- top left link under
Immunhistochemistry . Then scroll down to individual protein/ab hyperlinks.
Maybe you will find some suitable Abs.
NB: Sure you can use rat primaries on rat tissue, it just depends on whether
you are looking for NK cells
Can you submit an image?
I can imagine what this is but, no point until I see an image?
What are you doing that is different, if this is a new problem?
Curiously,
Carl
Carl Hobbs
Histology Manager
Wolfson CARD
School of Biomedical Sciences
Kings College London
Guys Campus
SE1 1UL
Tel: 020
I thought that I answered the original Q?
Imho, 3% H2O2 in distilled water is as good as any other recipe, given time
and concentration.
Sure, use a lower conc IF you achieve negative rbcs.
I use water because it is cheaper.
A lower concentration will take longerwhy bother??
Kill that
If you have tissue that has more peroxidase than usual, then extra time is
required.
Depends on the concentration of H2O2 used ;-)
A massive excess of substrate ( 3%) is good.
Carl
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
H202 mixed with Buffer here.(instead of aq.) That's OK too, right?
Right.
An addition to the misconceptions that Tim interestingly elucidates: some use
H2O2 in buffer because they think that , as the enzyme is performing a
physiologic catalytic reaction, it should be at a physiological pH, in
I made a mistakeI DO rehydrate before staining, as Tony pointed out.
I just don't use graded alcohols between the xylene and water ( x4 changes
74OP IMS).
Carl Hobbs
Histology Manager
Wolfson Centre for Age-Related Diseases
School of Biomedical Sciences
King's College London
Guys Campus
Why do some people use methanolic H2O2?
Why do people rehydrate after dewaxing?
Both are unnecessary, under usual conditions.
Methanol was used in the early days as a peroxidase blocker by itself.
The combination was devised as a belt and bracer method.
As you stated, you use aq H2O2 effectively.
Dear Gayle,
I would be interested to know why you are interested to know ;-)
Respectfully,
Carl
Carl Hobbs
Histology and Imaging Manager
Wolfson CARD
School of Biomedical Sciences
Kings College London
Guys Campus
SE1 1UL
Tel: 020 78486813
Fax: 020 78486816
020 78486813
Many thanks for the information, Ray.
carl
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
It may be that CD79a Ab is appropriate?
It works well on Pwax sections, after HIER.
I do not know the specificity of this Ag for B cells, so would be interested in
feedback
Check here for images : http://www.immunoportal.com/modules.php?name=gallery2
Carl Hobbs
Histology Manager
Wolfson CARD
Well, I have used all of the usual suspects over many years but, this one has
taken me by complete surprise!
If anyone can explain the rationale of the reaction, I would be most interested.
One for J Kiernan, I think?
Ah, yes: Sca/e: a chemical approach for fluorescence imaging and
Be careful: HIER may expose not only extracellular amyloid plaques but also
intracellular APP ( or...is it intracellular A beta ;-))
Eg: with 6E10 it is directed against an epitope found in APP and also A
beta: if you do Formic acid, only extracellular plaques are positive...after
Treat the tissue exactly as if it was human.
Sure, your anti CD68 may not work on pig but, it's worth a try.
LBH? Probably LDH. If so, better to measure serum levels?
Trypen blue? Prob. Trypan blue. It's a dye used for cell viability tests.
So, it seems that 2 and 3 are not IHC at all.
More
1) NO
2) Yes...at 4C
3) Sure, if somebody is around to do thisor you want to make somebody
go in and do it ;-)
carl
carl
Carl Hobbs
Histology Manager
Wolfson Centre for Age-Related Diseases
School of Biomedical Sciences
King's College London
Guys Campus
London SE1 1UL
Tel.020
Hey!
The GREAT Brian Lake was THE Man behind it all, imho.
Respect to Him.
Filipe, jest like Hiederman/Heyderman?, became famous because of the
people/technicians who did all of the work and...never got the credit ;-)
Carl Hobbs
Histology Manager
Wolfson CARD
Kings College London
020
Hey, Sara!
Congratulations.
Enjoy all them Hassles and Advantages in your NEW lab.
Best wishes,
Carl
NB: re Formalin fixation...one could argue that one should store such-fixed
specimens in 90% alcohol, after the appropriate Formalin fixation time?
OR...30% Sucrose: now, THAT is a PRESERVATIVE
I am trying to stain human glioma sections(as a positive control) using
Anti-NG2 antibody. The tissue that I have is Paraffin embedded and I appreciate
if somebody could send me the protocol for antigen retrieval ,antibody titer
and IHC protocol.
Let us know what Ab you are currently using (
I use Upstate's 06-570. Excellent for human, rat, mouse FFPW sections after
HIER.
I have no idea what amino acid is phosphorylated but, if you are wanting an Ab
against cells in mitosis only, this is the best, imho.
Carl
Carl Hobbs
Histology Manager
Wolfson Centre for Age-Related Diseases
I assume that you are using a biotinylated detection system?
If so, endogenous biotin labelling is specific, tho' NOT wanted ;-)
So, before you try variations, just buy a kit and see if your non-specific
immunostaining is due to endog. biotin ...follow their instructions by
pre-incubating in
Imhojust do it on your tissue and include a no primary control.
If the latter is clear, don't worry about MOM.
If it is not, then we can advise further.
You don't state what Ab/tissue/detection system you are using..
This would help a lot as protocols will vary according to the tissue
Used for quenching Formalin - induced fluorescence.
Carl Hobbs
Histology Manager
Wolfson Centre for Age-Related Diseases
King's College London
Tel.020 7848 6810
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
What species, V?
More info?
Them TGF ligand /receptors are as difficult as FGF/FGFr, imho.
respect,
Carlos.
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
I may have missed your explanatory post so, my apologies.
How do you know that your slides are dirty ?
Thanks,
carl
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
I have not yet found an anti mitochondrial Ab that works in mouse FFPE tissues,
so I am very interested.
However, the protein that Chemicon's MAB1273 is directed against survivesit
has worked very well for me in human FFPE sections after Citric acid HIER ( see
here for images:
Rene's comments are most appropriate.
Respect.
When I was in Clinical Histopath ( a technician) at two different well-known
London major Hospitals, it would be the technician who would be in the theatre
when renal bxs were taken.
EG; the person who took the biopsy would pass the specimen to
GFP can be detected directly in FFPwax sections if the expression level is high
enough.
Yo can easliy check this, using one section before you immunoprobe.
Sure, Ab detection will guarantee you a strong signal but, again, if the
exression of the protein is high enough.
I have come across
Following on from Paula Pierce's apposite answer: what DAB solution are you
using?
A DAB kit that may well contain metal enhancers?
carl
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
Many thanks for the replies regarding this reagent.
I delayed purchasing this as I was very skeptical: however, I can achieve a
1/5-10 fold dilution factor for my precious low affinity (? thus low dilution
factor) Abs.
I will not be using ImmPACT chromogen kit routinely as, for the vast
Has anyone tested this?
I would be very interested.
Ta
carl
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Thanks for adding a response.
However, I am a old Techie who does not understand what you are saying.
My apologies.
I use Alexa 488, 594 and also Hoechst to stain nuclei ( red, green , blue)
So, if I changed from MY Xcite system, to LEDs, can I be assured that those
three flourochromes I use
Re other IP chick images...I think that they were from using Millipore's
06-182.
I was concerned that blood vessels were also positive...
so I just dropped it.
Any ideas?
carl
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
Santa Cruz sc-101761.
I tested it on FFPW sections of rat brain sections, after Citric acid HIER and
it is very good ( if the results are specific, of course;-)
It is stated as being Human/Mouse/rat reactive.
I have two images posted here : http://www.immunoportal.com/index.php
for this Ab.
Your understanding is correct, imho.
I add a no primary control on my stABCpx-DAB run whenever I am testing new
tissues and assess any positivity that could be due to endogenous biotin.
You will quickly build up a knowledge of those tissues that automatically
require biotin - blocking. ( eg
Why a mouse Ab?
I have two anti-Cox-2 rabbit polys that seem to co-localise.
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
I am sure someone has a cunning combination!
I use Abcam's ab13970 chicken anti GFP in Pwax sections after HIER.
For me, it is very good
Image at Abcam or here : http://www.immunoportal.com/index.php
Good luck!
carl
___
Histonet mailing list
Are you using automated IHC-staining machinery, Naira?
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Apologies if I have not adhered to Histonet protocol: I have changed my Browser
from IE8 to latest Firefox and am not yet familiar!
I agree with Rene: stay with the same wells and give additional, longer washes.
Imho, even experienced users suffer section damage if they change wells.
Again, apologies if I have messed up with message delivery : Firefox from IE is
a learning curve after IE for many years.
Re your problem: only part that I reckon that is your problem is ONLY using
~20mins in an oven.
Leave for 2hrs at 60C, then stain.( overnight if you can is great for HEs)
I agree with Jason: have a look in image gallery here
http://www.immunoportal.com/
for an image of HES cells in mouse tissue, using V9 clone.
No personal experience with Human specific SMA but plenty of images
( ASMA) in IP gallery
Carl Hobbs
Histology Manager
Wolfson Centre for
I use a homemade DAB solution: I aliquot and freeze.
No problemo for 15 yrs, Patsy.
If you do not agree , please check out this site
http://www.immunoportal.com/index.php
All of my DAB images are developed using -20C DAB aliquots.
carlos
___
Histonet
Richard.I agree but I worry that
antibody 6E10 reacts to the abnormally
processed isoforms, as well as precursor
forms.
( my bold)
Does this mean that this Ab also demonstrates APP?
I must admit to confusion re this statement.
I would appreciate your further opinion.
respectfully,
Have a look in the image gallery here : http://www.immunoportal.com/index.php
I am interested to know why you want to avoid Formic acid AR.
In my XP, Formic acid exclusively exposes plaque-beta amyloid, using
appropriate Abs.
However, whether or not I am right, Citric acid ( or whatever your
http://www.immunoportal.com/index.php
carl
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Embedding frozen tissue in Pwax is very possible, Tomasz.
If your tissue has been frozen NOT using OCT, just place the frozen tissue into
acetone that has been cooled to -20C.
x3 changes of -20C acetone , then immediately place tissue into molten pwax x3
changes.
Agitate frequently, gently.
I
My sincere apologies...I uploaded the previous Histonet list because I forgot
to enter the appropriate re: in subject of email.
Contrite carl
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
I agree with Geoff re NFRed staining solution.
I have always had good results with home-made NFRed, if only for 2/4 months.
Much cheaper too.
Sure, try Neutral red: differentiate in water until you like the contrast then,
blot dry your section and immerse in Xylene after a time that you think has
Use a PC10 mouse clone for pwax sections: works very well on many species that
have been fixed in routine Formalin.
Imho, no special procedures reqd.
Sure, AR is best.
If you wish to check out this site: http://www.immunoportal.com/index.php
you might find further help?
Also , be very careful if
I use Chemicon's AB5320 anti NG2 ( 1/50-100) on mouse and rat pwax brain
sections. I need AR and use Citric acid pH6 in a microwaveable pressure
cooker.Standard stABCpxDAB detection.
Apart from the poor dilution factor, it's a very good reagent, imo.
Carl Hobbs
Histology Manager
Wolfson Centre
Hi Erin.
You don't state what the I.D. of the Chemicon Ab is, nor the species / tissue
prep. you are using.
I have compared Chemicon's AB1554 with Sigma's N3908 ( on rat and mouse
Formalin-fixed P.wax sections).
Also, Novocastra have an anti human NGFR ( gp75) : NCL-NGFR, which I have not
Just to try to help to make clear to those of us who get confused when people
talk about proof and % alcohol, I would recommend reading :
http://en.wikipedia.org/wiki/Proof_spirit
Proof is all about NOT getting a BANG from gunpowder ;-)
In UK Histopath/Histology labs, we traditionally use 64OP
101 - 156 of 156 matches
Mail list logo
