Hi Everyone
After years of putting it off, I finally took my ASCP HTL exam and passed it.
Huzzah!
Patrick.
Patrick Lewis
Research Associate II Bench
Seattle Childrens Research Institute
206-884-1115
CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for
the sole use
Hi everyone
I am going to take my ASCP HTL exam on the 21st of April
Any advice for studying?
I have been going over the sample online tests for both the HT and the HTL and
I bought the Michigan study guide.
Thanks
Patrick
Patrick Lewis
Research Associate II Bench
Seattle Childrens Resear
Hi everyone,
Has anyone experienced a problem with sigmas cc/mount solution.
I liberally cover my section with it to form a protective seal., But with my
current bottle it seems that when it dries it re-exposes the tissue and does
not form a protective layer.
I end up having to reapply it.
If I have an IHC where I am staining 2 slides from the same block, one with one
primary antibody and the other with a different primary antibody.
And one antibody's slides have high nonspecific background, but the other
antibody's slides have no background, Can I deduce that the background
sta
Hi Everyone
Thanks for your responses.
I am looking at cell surface markers,
Sorry I should have said.
Based on what I found out below:
Methanol is out, even though I agree that Methanol does enhance the effect of
H202 blocking.
(I suppose I could try it to see how much/if any epitope loss th
Hi everyone,
I seem to have a lot of endogenous Peroxide background staining in my FFPE IHC.
Human tonsil tissues, with some attached muscle.
I do a H202 block at 0.3% H202 in TBS pH 8.0 for 30 minutes.
Then I wash x3 with TBST 0.05% Tween20, pH 8.0
Then I serum block with 2% NGS in TBST fo
Hi Everyone,
I am seeing some red granular staining with my AEC substrate, and I am not sure
if its real, or Ice crystal artifact, or debris from the slide directly.
It's in several areas and is consistent between duplicates.
So can I rule out slide debris?
How deep would Ice crystals go in a
going
straight into buffer afterwards, but because I am using 100% I think that going
into liquid right after fixing is too much of a change and my tissues go BOOM.
Please help.
Patrick Lewis
Patrick Lewis
Research Associate II Bench
Seattle Childrens Research Institute
206-884-1115
CONFIDENTI
Hi Everyone,
I am still having issues with my IHCs with Acetone fixation.
If I fix in 100% Acetone, I get IHC staining, but my tissues are 50-90%
destroyed.
If I fix in 4% paraformaldehyde, or 10% NBF or (95% Etoh and/or Methanol with
Acetone) I lose the epitopes I either get no staining or
Hi Everyone.
When I fix my cryosections in acetone, I am using HPLC grade 99.9% for 10
minutes at -20C.
Would the Histology grade 99.5% be less damaging to them?
Higher H20 content, i.e. less than 99.5% apparently is also very bad.
With the HPLC grade I often get tissue damage, the tissue al
Hi Everyone,
Can anyone recommend a good Cryostat to buy for use with Human Tissues.
I'd like to look for a reasonably priced model.
Also, I'd consider paying extra for a model that is designed for human tissues
so it has disinfection ability or is easier to disinfect than older models.
I'd als
Hi everyone,
Does anyone know who I would contact about servicing an old CM3000 Cryostat.
This would be in the Seattle area and we have no service contract.
I am going to call Leica tomorrow, but if anyone has information about
repair/servicing that would be helpful.
Thanks
Patrick.
CONFID
H everyone,
We have been using this old CM3050 cryostat for our Frozen OCT blocks.
I am cutting at 5 uM.
My issue is that when I use the hand rotator to cut my sections it doesn't seem
to advance with each rotation. It can take as many as 4 rotations to go from
one section to another.
What w
After having a lot of problems using 100% Acetone as a fixative, I am going to
try 4% paraformaldehyde in DPBS pH 7.4 for fixation of slides cut from fresh
tissues snap frozen in OCT.
My plan is to cut the slides at 5 or 6 uM and dry them for 2 hours in the hood,
then fix them in the 4% p-f f
Hi everyone.
If you have several OCT blocks in which the Tissue has fallen out and to want
to salvage them for IHC. What is the best way to do so to minimize freeze-thaw
damage.
These samples were harvested and then were probably snap frozen in Isopentane
surrounded by dry ice and stored at
Hi Everyone.
I make my Frozen blocks by Immersing the cryo molds in a metal container (Looks
like a larger flat base ½ coke can) filled with Isopentane solution. That
container is then surrounded by Dry Ice. We need a new metal container. Does
anyone know where I could buy one?
I don't rea
Hi everyone.
With Snap frozen tissue in OCT. If I cut sections and fix them in cold acetone
for 10 minutes, (Is 2 minutes vs 10 minutes going to cause any issues?)
Is it better to dry them and then put them in DPBS or just put them straight
into DPBS?
If I changed the fixative to 10% NBF woul
Thanks everyone,
I just posted 2 images to histo search. IS there a way to post up to 5 pics at
once? It looked like it suggested that you could post up to 5 pics as a group
but I didn't see HOW to do it.
Anyway, I have one post of outside of the tissue area, and one post of the
tissue are
Hi everyone,
What is a good way to post pictures of slides for you guys to look at?
I had a weird black precipitate in one of my IHCs and I'd like for you guys to
look at them and see if you recognize what this artifact is?
Thanks
Patrick.
PS: I tried uploading them as attachments but the siz
Hi everyone.
I was staining a FFPE macaque liver tissue slide with hematoxylin the other
day, and it had an odd appearance.
All the cells stained the usual way with the blue nuclear staining that you
expect with hematoxylin.
But a few randomly scattered individual cells throughout the tissue s
Hi everyone,
Does anyone know where I can get replacement parts for a Stovall Belly Dancer
bench top shaker?
One of the 4 tubing legs has snapped near the base. I might be able to
jury-rig it with wire and duct tape, but it does have screws at each end of the
tubing so It looks like the tubin
Hi Everyone,
Can anyone tell me the cheapest place to get True North Slide boxes (100 slides
purple box)
VWR has them for about $33.00 EACH.
We get a shipping discount with VWR but there has got to be a cheaper supplier.
Baring that, can anyone recommend a good 100 slide storage box. Prefera
Hi everyone
Can someone recommend a good place to buy Copland jars.I want the glass
jars with the plastic circle top screw on lid.
I only want about 4 of them and cost is an issue.
Thanks
Patrick.
CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for
the sole u
Hi Everyone.
I am trying to troubleshoot my IHC on frozen sections.
My sections are human tonsil at 7 uM. On charged Superfrost slides.
They are stored at -80 after drying for 1 hour.
When I use them for IHC, I take them out of the -80 and let them air dry for 1
hour before placing them in co
Hi everyone
I am having some trouble with my IHC on frozen OCT sections.
The antibody/secondary/AEC works great on my Epitope retrieved paraffin
sections.
However,
When I try a similar IHC with frozen OCT blocks. Different samples however,
but still should be positive.
I get lots of nonspec
Hi everyone,
Has anyone had any problems with the Immedge Pap pen from Vector. Cat # H-4000
It gives a nice wide barrier, but for some reason the barrier is disintegrating
on our slides so I get holes in the lines I draw.
I have had a similar problem before, usually because the slide wasn't dry
Hi everyone,
Can someone recommend a good pen to use to label cryo tubes.The VWR pens we
have been using are apparently discontinued.
I would like something that writes as fine as an ultrafine sharpie, but is less
likely to smear and is more long lasting, and solvent tolerant.
Thanks
Patr
Hi Everyone,
Just out of curiosity, what sort of nanodrop values do you typically get with
DNA extractions from paraffin sections.
I usually get around 100 ng/uL, depending on tissue size, and the number of
churls I take. Around 80% are 100 ng to 200 ng/uL.
Rarely, I get as high as 1000 ng/uL,
HI everyone,
Does anyone know where to get 5E1 mouse monoclonal antibody directed against
HHV-7 (pp85 protein complex)
We tried two suppliers but their product has been discontinued and now we can
not seem to find it anywhere.
Thanks
Patrick.
CONFIDENTIALITY NOTICE: This e-mail message, inc
Hi everyone, can someone recommend a good storage method for Frozen tissue
blocks made from cryomolds? We have about 200 blocks.
Currently we have them stored freezer boxes in bags with some wet ice at -70.
It might be better if we had plastic tray bins to put the bags in, but I have
not been
Hi Everyone,
Can anyone recommend a good Rabbit Isotype Control Antibody IgG1, to use as a
negative control for my Polyclonal Rabbit antibodies.
I am looking at Biocompare and GenTex has one for about $220.00 for 100 ug.
GTX47478
But if someone has one they've used and works well, I'd be inter
Hi Everyone,
Thanks for the response earlier.
Does anyone any advice on EBER ISH Kits? Specifically, if they had better
results, or less problems with one brand vs another?
Right now I am considering both Novacastra's kit for 970.00 and Vector Labs kit
for 905.00
Has anyone used these kits? H
Hi Everyone,
I am going to do some ISH for EBER on FFPE sections, and I have some concerns
on the logistics.
The kits I am looking at are for 50 rxns, and it seems like they expect to use
20 uL probe per rxn.
My sections are rather large, covering about 2/3 of a standard slide.
How many uL w
Hi everyone,
That should read,
Would anyone able to recommend a good antibody to stain for only mast cells in
human tonsil for IHC.
Thanks in advance.
Patrick Lewis
CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for
the sole use of the intended recipient(s) and
Hi everyone.
Would anyone be able anyone recommend a good antibody to stain mast cells in
Human Tonsil for IHC?
These would be in FFPE (Formalin fixed paraffin embedded sections)
With secondary antibody hopefully being HRP labeled.
Substrate would most likely be DAKO AEC.
Thanks
Patrick Lewi
So what I'm gathering from all this.
Thanks by the way for the discussion.
Methanol is unnecessary for the H2O2, reaction, but it can make it slightly
faster since both methods will be working in concert. If you have tissue that
has more peroxidase than usual, then extra time is required.
Al
Hi everyone,
Can someone refresh/enlighten me as to why Methanol is used in H2O2 blocking.
Let's assume that I take my slides through the xylene/ethanol steps and that
after the 70% etoh wash step, they are now in TBST.
I could see adding methanol if you were doing the H2O2 blocking as part of
Hi everyone,
I have been using DAKO Kits for HRP anti rabbit and HRP anti Mouse and AEC
substrates.
I love DAKO, but their kits and reagents are expensive.
Can anyone offer an alternative for HRP labeled anti (Rabbit/Mouse) antibodies
and AEC substrate kits.
I do all my IHC manually by hand.
Dear Histonetters
Yes I should have said, I'll be doing IHC for viral proteins with both
polyclonal (rabbit) and monoclonal (Rat/Mouse) primary antibodies and
HRP-labeled secondary antibodies with DAKO AEC Substrate.
Patrick Lewis
Research Associate II-Bench| Infections and Prematurity
Hi guys,
Can anyone recommend a good decal solution. I have some bone marrow and
trachea tissues for paraffin sectioning and I want to decal them.
Thanks
Patrick
Patrick Lewis
Research Associate II-Bench| Infections and Prematurity
Seattle Children's Research Institute
206-884-111
Hi Histonetters
When I soak my slides in xylene to remove the coverslip, do I then need
to rehydrate them by washing them in 100% etoh,95% etoh,70% etoh, and
then H20.
And then drying and re applying permount?
Or can I go straight from xylene to drying to re-coversliping with
permount?
If I want to remove a cover slip that is coversliped with permount. Do
I need to use xylene to get rid of the semi hardened permount gunk?
Also, If I am heating paraffin sectioned slides overnight at 37C to
adhere them what happens if I over cook them by leaving them at 37C for
more than one
Hi histonetters
Has anyone got more info on the superfrost plus poor quality issue?
Are there specific lot # that are going to be recalled?
Is there a way to find out if any of our boxes of slides are likely to
be bad/poor adherence?
I just started doing some IHCs and I am crossing my finger
Hi Histoneters
Can someone explain sucrose cryoprotection to me.
Why/when is it necessary?
Tissue types that require it?
Potential benefits/problems with its use or not use?
Thanks.
Patrick
Patrick Lewis
Research Associate II-Bench| Infections and Prematurit
I have some questions about Fixing tissues for paraffin embedding when
the ultimate purpose will be looking for viral antigens and cell surface
markers.
We have some monkey tissue that has been in NBF for over a year. and we
are just now processing them and embedding them in to paraffin.
We
Has anyone used sanosil as a disinfecting agent? Could it be used to
disinfect a cryostat? Is it possible to use its fogging method to make
sure that all the crevices within a cryostat are covered with
disinfectant. Would sanosil affect future slides that are made in the
cryostat that has residu
When I was cutting my block I noticed some ribboning of the tissue. Is
there a known cause (or several) for this. I am wondering if my
microtome blade was too tight or too loose or if my block froze poorly,
or if I just needed to reorient my block . I was cutting 7 um at -20.
Its human tonsil,
Hi everyone,
I cut my first frozen sections, human tonsil, and I have stored them at
-70. When I take them out to use, should I bring them to -20C and fix
them right away? Or should I bring them to room temp and then pap
pen/wash/block/wash them then fix them before adding my primary
anti-bod
Hello everyone,
My name is Patrick Lewis and I just started working for Children's Hospital in
their Research Department.
It's been a long time since I've done any Immunohistochemistry, (~10 years),
but in this new job, I will be doing a lot of it with both paraffin and frozen
sections t
49 matches
Mail list logo