Re: [Histonet] Invitrogen A6455 rabbit anti-GFP dilution for

[Hobbs, Carl]

 IF using Alexa - conjugated secondaries ( indirect IF) should give you the 
same results as a std  stABCpx-DAB detection, for any given dilution of  
primary Ab.
However, if you are using enhanced DAB.sure, the primary Ab will have to be 
less dilute when visualising with an  indirect IF method.
So, do one run with your primary at 1/5K, 1/10K, 1/20K ( on three sequential 
sections, of course) ...detecting with, for eg, Alexa anti Rabbit 594 at 1/1K.

Carl Hobbs FIBMS
Histology and Imaging Manager
Wolfson CARD
Guys Campus, London Bridge 
Kings College London
London
SE1 1UL
 
020 7848 6813

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[Histonet] RE: Waterbaths

[Tony Henwood (SCHN)]

Hi LeAnn,

We empty the waterbaths each afternoon and add fresh warm water in the morning 
(we do not need to use distilled water - fortunately Sydney has good clean 
water, as well as high taxes, high house prices, etc etc!!)

We clean with warm soapy water.

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Laboratory Manager & Senior Scientist 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Leann M. Murphy
Sent: Tuesday, 6 August 2013 3:02 AM
To: Histonet@lists.utsouthwestern.edu
Subject: [Histonet] Waterbaths

Just wondering if everyone leaves the distilled water in their water baths 
overnight or replaces the water first thing in the morning?  Also, what do you 
use to clean your water baths?

LeAnn Murphy
Technical Specialist Aultman Hospital
lmurp...@aultman.com

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[Histonet] RE: Slide dehydration After Staining Tissue Falling Off

[Goins, Tresa]

After the detergent treatment, rinse well in distilled water and dry in the 
oven.
Dip in clearant and coverslip.

We do not treat our developed IHC slides in alcohols or clearant due to 
potential loss of some labeling - not DAB.

Tresa

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Christopher 
Jacobs
Sent: Monday, August 05, 2013 11:53 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Slide dehydration After Staining Tissue Falling Off

Histonetters,

Recently my lab has been experiencing a significant occurrence of tissue 
falling off our IHC slides. We are looking at a variety a factors and I have 
been watching how some technicians dehydrate their slides after staining. We 
are running Ventana IHC stainers, so we remove the oil based liquid coverslip 
in a weak solution of Dawn and then run the slides down alcohols into xylene. 
Some technicians are very aggressive in their agitation of the slides. It is 
almost comical how much sloshing around in the reagents they do. How can I get 
these people to ease up? Does anyone have any protocols on how many dips it 
takes in each reagent to properly dehydrate slides?

Also, some of the other issues we are looking at causing tissue falling off the 
slides are; tapping slides to remove water, not double-dipping slides, lotion 
on hands, over temperature, and the brand of charged slides we are using. Can 
anyone think of any other factors to look at?

Thanks!

"CJ" Christopher P. Jacobs, HT QIHC(ASCP)



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RE: [Histonet] MART1 Stain Frozen Section Samples

[Elizabeth Chlipala]

David

I can't help you out with the MART 1 IHC on frozen but DO NOT store your 
positive control block in the cryostat it should be stored at -70C if possible. 
  We store our frozen section control blocks at -70C.  Once the block has been 
sectioned we cover the tissue with a thin layer of OCT and then store in tiny 
plastic bags in our -70C freezer.  If you think you are going to be using these 
a lot I would store on the chuck that way you will not loose too much tissue 
when you have to recut, or you can cut a bunch of control slides at one time.  
The issue with that is that they should not be stored all in one box, what you 
want to avoid is numerous freeze/thaw cycles for the control slides.  We cut 
the frozen sections let air dry at room temp and then place them multiple in 
slide boxes, we store what we need for a particular run in one box.  In the box 
we place a small bag of indicator silica gel (this will absorb any excess 
water, Silica gel, 6-16 mesh indicating is what we use) and seal the slide box 
in a plastic food seal bag using a food sealer, place in -70C.  We take out of 
the freezer the night before and place at room temp prior to staining.  This 
seems to work well for us.  If we are cutting just control slides we use the 5 
slide mailers, or you could even use the plastic two slide holders that are 
flat, place the silica gel on top of the plastic holder and seal both together. 
 We use nylon tissue biopsy bags to place the silica gel in and we just staple 
the bag shut.

Good Luck

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 881-0763 cell
(303) 682-9060 fax
l...@premierlab.com

Ship to address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

From: histonet-boun...@lists.utsouthwestern.edu 
[histonet-boun...@lists.utsouthwestern.edu] On Behalf Of David Anderson 
[david.w.ander...@vanderbilt.edu]
Sent: Monday, August 05, 2013 1:06 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] MART1 Stain Frozen Section Samples

Hello everyone,

I am currently working in the MOHS Laboratory for Vanderbilt
Dermatology and the physicians are wanting to start performed MART1
staining on the frozen tissue samples.  Is there anyone out there that
also works in a MOHS laboratory and has protocols already in place
that would like to share their protocols?  Also, the positive control
tissue...how do you store it until use...in the cryostat? Just any
information or insight that you have will be greatly appreciated.
Thank you my fellow Histotechs!!!

Sincerely,
David Anderson, HT


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[Histonet] MART1 Stain Frozen Section Samples

[David Anderson]

Hello everyone,

I am currently working in the MOHS Laboratory for Vanderbilt  
Dermatology and the physicians are wanting to start performed MART1  
staining on the frozen tissue samples.  Is there anyone out there that  
also works in a MOHS laboratory and has protocols already in place  
that would like to share their protocols?  Also, the positive control  
tissue...how do you store it until use...in the cryostat? Just any  
information or insight that you have will be greatly appreciated.   
Thank you my fellow Histotechs!!!


Sincerely,
David Anderson, HT


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RE: [Histonet] EM tissue processors

[Morken, Timothy]

Thomas, I forgot to answer your other questions. The RMC and Leica conventional 
processors are similar in function and do a fine job of processing samples. We 
had the RMC for many years before getting the Leica. We use the RMC as a 
back-up now. 

The RMC will hold more samples due to a much larger vial. It is reliable, but 
repairs require you send the unit to the factory in Tucson. They are very 
helpful but do not have a field staff. The Leica will do on-site repairs and 
offers service contracts. RMC does not have service contracts.  I have only had 
to send the RMC in for repairs once after almost 8 years of use. It had been 
pretty beat up and since we got it back it has performed flawlessly. 

So far the Leica can handle what we need to do  a maximum (so far )of 12 
samples in one batch. 

Both are easy to use and to program. Once they are programmed they are simple 
to start. Both will heat and cool the sample, though I think the Leica is more 
uniform since the cooling/warming chamber completely encloses the vial. The RMC 
uses a Piezo block to cool/warm one side of the sample. 

The Leica goes through the routine a little faster because the reagent 
change-over is a little faster. The RMC has a cap on each vial that has to be 
removed (automated) while the Leica does not (vial caps are attached to the 
lid). It is about 15 minutes on a 3 hour schedule, so not really significant. 
(depends on the number of vials your protocol has). One difference - since the 
Leica has the vial covers attached to the lid you cannot take the lid off. With 
the RMC, if you have inside a fume hood you can take the lid off for more 
convenient access (Actually, with the RMC you cannot use the lid in a fume hood 
because it attached at the back of the unit and would be blocked from opening). 

The RMC fits in a regular fume hood. The Leica does not - it is too long front 
to back, and you could not anyway because the lid must be attached and will not 
lift because the backside would be hard against the back of the hood. However, 
Both have covers, exhaust fans and hoses to vent into a fume hood. Right now we 
have the RMC in the hood (a 6-foot hood) and the Leica outside. We have not had 
any fume problems with the Leica (we used to have the RMC Outside the hood and 
did not have any problems with it either. We had a new, larger hood installed 
and put it in there because it would not fit on the bench next to the Leica).

As I mentioned, there is no significant difference in cost of the reagents, and 
both units perform fine. We got the Leica only because we wanted a back-up and 
wanted to get a more modern  instrument. Our validation showed they perform 
identically to process tissue to our specs.

Tim

-Original Message-
From: Morken, Timothy 
Sent: Monday, August 05, 2013 10:37 AM
To: 'Tom Strader'; histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] EM tissue processors

Thomas,

We use the RMC and the Leica TP-EM (conventional) EM tissue processors, They 
are similar in equipment cost and vial pricing (in fact, I believe the vials 
are made in the same facility) about 15K. Our consumable costs for both units 
(buffers, osmium, solvents, resin) comes to about $13.00 per batch, and works 
out to $2.57 per case (about 5 cases per day average).  We re-use the non-resin 
vials for a week each to save a bit. For us the equipment payback is about 166 
working days (saves about 2 hours tech time per day - changing reagents 
manually).  

The Leica MW processor is about $50K, so the payback will be about 3.5 times 
longer. The reagent cost for manual vs any automated system won't be much 
different and are reagents are so cheap it is not a significant cost compared 
to labor costs.

We do mostly kidneys, but also muscle (6 cases/wk) liver, heart, blood, and 
some tumor. We run all samples on a 3-hour processing schedule so we can get 
outside cases done the same day they come in. 


Tim Morken
Supervisor, Electron Microscopy/Neuromuscular Special Studies Department of 
Pathology UC San Francisco Medical Center



-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Tom Strader
Sent: Thursday, August 01, 2013 9:43 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] EM tissue processors

Hi,
I'm comparing operating costs relative to features/benefits of automatic EM 
tissue processors (e.g. Leica, RMC, Lynx,.) with or without microwave and was 
wondering if anyone else had already done the same. I'd like to compare total 
costs per specimen and relative advantages/disadvantages of each system. I'm 
looking at the cost of consumables, reagents and labor separately and am also 
interested in reliability, efficiency and ease-of-use.
I'd greatly appreciate it if anyone has information they can share.
Feel free to contact me at the address below if you have questions.
Thanks!
Best regards,
Tom
 
Thomas E. Strader, MS
He

RE: [Histonet] Hardware from surgery

[Dessoye, Michael J]

We retain for two years post sign-out.  If the patient is a minor, then
until their 18th birthday plus two years.

Michael J. Dessoye, M.S. | Histology Supervisor | Wilkes-Barre General
Hospital | An Affiliate of Commonwealth Health |
mjdess...@commonwealthhealth.net | 575 N. River Street | Wilkes Barre,
PA 18764 | Tel: 570-552-1432 | Fax: 570-552-1526 


-Original Message-
From: anita dudley [mailto:azdud...@hotmail.com] 
Sent: Friday, August 02, 2013 12:33 PM
To: Histonet@lists.utsouthwestern.edu
Subject: [Histonet] Hardware from surgery

What do others do with hardware from surgery?  Do you hold on to it for
a longer period of time because of a possible legal case?   Thanks so
much, everyone have a great weekend!!

 

Anita Dudley

Providence Hospital

Mobile, Al. 36695
  
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

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[Histonet] Slide dehydration After Staining Tissue Falling Off

[Christopher Jacobs]

Histonetters,

Recently my lab has been experiencing a significant occurrence of tissue 
falling off our IHC slides. We are looking at a variety a factors and I have 
been watching how some technicians dehydrate their slides after staining. We 
are running Ventana IHC stainers, so we remove the oil based liquid coverslip 
in a weak solution of Dawn and then run the slides down alcohols into xylene. 
Some technicians are very aggressive in their agitation of the slides. It is 
almost comical how much sloshing around in the reagents they do. How can I get 
these people to ease up? Does anyone have any protocols on how many dips it 
takes in each reagent to properly dehydrate slides?

Also, some of the other issues we are looking at causing tissue falling off the 
slides are; tapping slides to remove water, not double-dipping slides, lotion 
on hands, over temperature, and the brand of charged slides we are using. Can 
anyone think of any other factors to look at?

Thanks!

"CJ" Christopher P. Jacobs, HT QIHC(ASCP)



CONFIDENTIALITY NOTICE: This message and any attachments are for the sole use 
of the intended recipient(s). This message is confidential and may also be 
privileged. If you are not the intended recipient, please contact the sender 
immediately, delete the contents of this message and do not use it for any 
purpose.

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RE: [Histonet] EM tissue processors

[Morken, Timothy]

Thomas,

We use the RMC and the Leica TP-EM (conventional) EM tissue processors, They 
are similar in equipment cost and vial pricing (in fact, I believe the vials 
are made in the same facility) about 15K. Our consumable costs for both units 
(buffers, osmium, solvents, resin) comes to about $13.00 per batch, and works 
out to $2.57 per case (about 5 cases per day average).  We re-use the non-resin 
vials for a week each to save a bit. For us the equipment payback is about 166 
working days (saves about 2 hours tech time per day - changing reagents 
manually).  

The Leica MW processor is about $50K, so the payback will be about 3.5 times 
longer. The reagent cost for manual vs any automated system won't be much 
different and are reagents are so cheap it is not a significant cost compared 
to labor costs.

We do mostly kidneys, but also muscle (6 cases/wk) liver, heart, blood, and 
some tumor. We run all samples on a 3-hour processing schedule so we can get 
outside cases done the same day they come in. 


Tim Morken
Supervisor, Electron Microscopy/Neuromuscular Special Studies
Department of Pathology
UC San Francisco Medical Center



-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Tom Strader
Sent: Thursday, August 01, 2013 9:43 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] EM tissue processors

Hi,
I'm comparing operating costs relative to features/benefits of automatic EM 
tissue processors (e.g. Leica, RMC, Lynx,.) with or without microwave and was 
wondering if anyone else had already done the same. I'd like to compare total 
costs per specimen and relative advantages/disadvantages of each system. I'm 
looking at the cost of consumables, reagents and labor separately and am also 
interested in reliability, efficiency and ease-of-use.
I'd greatly appreciate it if anyone has information they can share.
Feel free to contact me at the address below if you have questions.
Thanks!
Best regards,
Tom
 
Thomas E. Strader, MS
Heartland Biotech | Madison, WI, USA | 608-770-7649 | 
 www.heartlandbiotech.com 
tom.stra...@heartlandbiotech.com
 
 
 
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RE: [Histonet] Waterbaths

[McAnn, Sherrian]

Sorry I forgot to say that I like to use dish soap..namely dawn...but
almost any laboratory glassware soap would probably do the trick.

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Leann M.
Murphy
Sent: Monday, August 05, 2013 12:02 PM
To: Histonet@lists.utsouthwestern.edu
Subject: [Histonet] Waterbaths

Just wondering if everyone leaves the distilled water in their water
baths overnight or replaces the water first thing in the morning?  Also,
what do you use to clean your water baths?

LeAnn Murphy
Technical Specialist Aultman Hospital
lmurp...@aultman.com

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RE: [Histonet] RE: Waterbaths

[McAnn, Sherrian]

I  wash mine with hot soapy water and a soft green scrubby to remove the
day's paraffin and bacteria (I'm sure it grows some).  Then squirt a
little absolute in it and wipe around to help dry it .  We have a
morning person who fills all water baths every morning. Personally I
don't like the idea of letting water sit overnight as I feel that dust
can fall into it.  Also I feel like it could be a good  breeding ground
for bacteria. This is just my personal opinion

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Sanders,
Jeanine (CDC/OID/NCEZID)
Sent: Monday, August 05, 2013 12:05 PM
To: Leann M. Murphy; Histonet@lists.utsouthwestern.edu
Subject: [Histonet] RE: Waterbaths

I empty mine each day and dry it. Mine does not have a glass insert or I
would run it through the dishwasher so instead I wash it every few days
with warm, soapy water.

Jeanine H. Sanders
Centers for Disease Control and Prevention Infectious Diseases Pathology
Branch
404-639-3590
j...@cdc.gov


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Leann M.
Murphy
Sent: Monday, August 05, 2013 1:02 PM
To: Histonet@lists.utsouthwestern.edu
Subject: [Histonet] Waterbaths

Just wondering if everyone leaves the distilled water in their water
baths overnight or replaces the water first thing in the morning?  Also,
what do you use to clean your water baths?

LeAnn Murphy
Technical Specialist Aultman Hospital
lmurp...@aultman.com

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[Histonet] RE: Waterbaths

[Sanders, Jeanine (CDC/OID/NCEZID)]

I empty mine each day and dry it. Mine does not have a glass insert or I would 
run it through the dishwasher so instead I wash it every few days with warm, 
soapy water.

Jeanine H. Sanders
Centers for Disease Control and Prevention
Infectious Diseases Pathology Branch
404-639-3590
j...@cdc.gov


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Leann M. Murphy
Sent: Monday, August 05, 2013 1:02 PM
To: Histonet@lists.utsouthwestern.edu
Subject: [Histonet] Waterbaths

Just wondering if everyone leaves the distilled water in their water baths 
overnight or replaces the water first thing in the morning?  Also, what do you 
use to clean your water baths?

LeAnn Murphy
Technical Specialist Aultman Hospital
lmurp...@aultman.com

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[Histonet] Waterbaths

[Leann M. Murphy]

Just wondering if everyone leaves the distilled water in their water baths 
overnight or replaces the water first thing in the morning?  Also, what do you 
use to clean your water baths?

LeAnn Murphy
Technical Specialist Aultman Hospital
lmurp...@aultman.com

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[Histonet] Invitrogen A6455 rabbit anti-GFP dilution for immunofluorescence

[Bass, Caroline]

Hi Everyone,

I use Invitrogen/Life Technologies/Molecular Probes' antibody for GFP staining 
in PFA fixed rat brains. It works quite well in my hands at a concentration of 
1:20,000 for DAB staining. However, I have need of immunofluorescence now and 
don't have a good starting point. The product sheet isn't very helpful and 
Invitrogen says to use the same Ab concentration which seems crazy. Almost 
everything I see shows a lower dilution with IF. 

Does anyone have experience with this particular antibody in the brain? If not, 
what's a good rule of thumb when trying to convert an antibody from DAB to IF? 
What would you suggest in terms of dilution?

Thanks!

Caroline



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[Histonet] Re: Air bubbles in coverslipper

[Teri Johnson]

Hi Anna,

Have you watched the slides get coverslipped and noticed it blowing air bubbles 
when it is dispensing the mounting medium? If no, then that is not where your 
problem is occurring.  Has your mountant gotten thick and is more viscous than 
when new?  What is your level of xylene in your bath? If it's low it can trap 
an air bubble at the very top of the slide when it finishes dropping the 
coverslip.

Teri Johnson
Manager, Histology
GNF - San Diego, CA
858-332-4752

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[Histonet] Wednesday

[Mack, Sarah]

I will not be here on Wednesday. 


Sarah Mack
University of Rochester Medical Center
Center for Musculoskeletal Research
Histology, Biochemistry, and Molecular Imaging Core
601 Elmwood Avenue
Box 665
Rochester, NY 14642
(585)-273-3901
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RE: [Histonet] Re: Portable fans in the lab

[joelle weaver]

I remember the issues in Tennessee. It would be nice if they considered the 
work environment for you and others. I have noticed that it seems to also 
depend on the organizational policies (there/where ever you),  are as far as 
what electrical devices are permitted. When I have worked elsewhere, using a 
fan for drying was not an issue so long as it was checked for electrical 
safety. 




Joelle Weaver MAOM, HTL (ASCP) QIHC
 
> Date: Sun, 4 Aug 2013 14:21:37 -0400
> From: rsrichm...@gmail.com
> To: histonet@lists.utsouthwestern.edu
> Subject: [Histonet] Re: Portable fans in the lab
> 
> I've attempted to use a portable fan to dispel formaldehyde fumes in a lab
> with no effective ventilation (not required in Tennessee) and was told by
> management they were prohibited. OSHA seems not to understand that
> pathologists and histologists are people too.
> 
> Bob Richmond
> Samurai Pathologist
> Maryville TN
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RE: [Histonet] Portable fans in the lab

[joelle weaver]

No arguments to anyone's tips about water on/within/underneath cut sections. I 
just use the fan though to help dry the hand coverslipped slides since I don't 
have a coverslipper.  In a hospital setting, they usually have to do the 
electrical check and provide the biomed sticker & certification. I have also 
seen hair dryers used. 




Joelle Weaver MAOM, HTL (ASCP) QIHC
 
> Date: Sun, 4 Aug 2013 12:31:40 -0400
> From: bakevicto...@gmail.com
> To: rjbu...@yahoo.com
> Subject: Re: [Histonet] Portable fans in the lab
> CC: histonet@lists.utsouthwestern.edu; paintedsplas...@yahoo.com
> 
> Hi
> Rene is correct about standing them up right to drain, but if you are using
> charged slides water gets trapped underneath the sections because of the +
> charge of the slide itself.  If you see a bubble on the last section or
> around  sections on a ribbon you can release the water by just pin pricking
> them on the edge with a needle point or the tip of a fine
> tipped forceps. Tap the section and the water will drain away from the
> tissue.
> Joelle is on target with use of a fan if your lab has an increased or high
> humidity level as this will also affect the water retention on a slide.
> 
> 
> 
> 
> 
> 
> On Sun, Aug 4, 2013 at 11:50 AM, Rene J Buesa  wrote:
> 
> > In my experience the best procedure is to place the slides with the
> > sections just "fished" from the water bath in a vertical position on the
> > short edge (1 plg).
> > By doing so the water that may be under the section will come out and
> > after about 5 minutes the slides can be placed in the rack and either go to
> > the oven to dry before manual staining or directly to the autostainer
> > heating station.
> > If you blow air over a section that is in horizontal position any water
> > trapped under the section will remain there and can cause the artifact
> > known as "nuclear bubbling".
> > René J.
> >
> >
> > 
> > From: Jeanne Clark 
> > To: "histonet@lists.utsouthwestern.edu"  > >
> > Sent: Saturday, August 3, 2013 5:34 PM
> > Subject: [Histonet] Portable fans in the lab
> >
> >
> > Anyone using portable fans in Histology to facilitae drying of parafin
> > sections on slides prior to drying in oven?
> >
> > Jeanne
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