Re: [Histonet] Faded H tissue section

[jayalakshmy p.s via Histonet]

Thanks, I'll check it out.

On Fri, Nov 10, 2023, 6:58 AM Tony Henwood  wrote:

> You could treat the decoverslipped section in 1% periodic acid (same as
> used in the PAS technique)  for 30 minutes prior to H staining. This
> might improve the H staining.
>
>
>
> I believe Lee Luna suggested this but for the life of me, I can’t find the
> reference!
>
>
>
> Regards,
>
>
>
> Tony Henwood MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) (Retired)
>
> Principal Scientist, the Children’s Hospital at Westmead (Retired)
>
> Adjunct Fellow, School of Medicine, University of Western Sydney.
>
>
>
> *From: *jayalakshmy p.s via Histonet 
> *Sent: *Friday, 10 November 2023 3:43 AM
> *To: *histonet@lists.utsouthwestern.edu
> *Subject: *[Histonet] Faded H tissue section
>
>
>
> Hello,
> I would like to know how to effectively restain a faded H tissue section.
> The color becomes dull when re stained. Somebody please advise.
> Prof. Jayalakshmy. P S
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>
>
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[Histonet] Faded H tissue section

[jayalakshmy p.s via Histonet]

Hello,
I would like to know how to effectively restain a faded H tissue section.
The color becomes dull when re stained. Somebody please advise.
Prof. Jayalakshmy. P S
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Re: [Histonet] Clarification about Immunohistochemistry

[jayalakshmy p.s via Histonet]

Thank you for the clarification and the answers of all others

On Wed, Apr 13, 2022, 2:05 AM Tony Henwood (SCHN) <
tony.henw...@health.nsw.gov.au> wrote:

> "Bond wash" is the propriety buffer used in the Bond Immunostainer.
>
> *Regards*
> *Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)*
> *Principal Scientist, the Children’s Hospital at Westmead*
> *Adjunct Fellow, School of Medicine, University of Western Sydney*
> *Tel: 612 9845 3306*
> *Fax: 612 9845 3318*
> *Pathology Department*
> *the** children's* *hospital* *at westmead*
> *Cnr Hawkesbury Road and Hainsworth Street, Westmead*
> *Locked Bag 4001, Westmead NSW 2145, AUSTRALIA*
> --
> *From:* jayalakshmy p.s 
> *Sent:* 12 April 2022 22:01
> *To:* Tony Henwood (SCHN) 
> *Cc:* histonet@lists.utsouthwestern.edu  >
> *Subject:* Re: [Histonet] Clarification about Immunohistochemistry
>
> Thank you so much Susan and Tony. Let me prepare the reagents 
> To Susan- can you please elaborate on the steps of the giemsa method.
> To Tony Henwood - please tell what is meant by "bond washing"
> Thanks
> Dr. Jayalakshmy
>
> On Tue, Apr 12, 2022, 9:58 AM Tony Henwood (SCHN) <
> tony.henw...@health.nsw.gov.au> wrote:
>
> Hi there,
>
> I have had good results with Giemsa-like counterstaining (Stefanović  et
> al 2013, Ravishankar et al 2016)  :
>
> Azure blue, when substituted for hematoxylin as a counterstain in
> immunostain preparation, has been used to help differentiate melanocytes
> from melanophages. Azure blue preferentially stains cytoplasmic melanin
> granules blue-green, whereas melanocytes are highlighted by brown DAB
> chromogen. Melanophages, which contain melanin and lack melanocytic
> determinants, appear clear with blue-green granules in the cytoplasm
> (Hillesheim et al 2011).
>
> After the slides were bond washed for 4min and rinsed in distilled water,
> they were stained with a mixture of the following solution: 100 mg of Azure
> blue (Sigma) in 4 ml of distilled water. Solution 2 was prepared as
> follows: 0.6ml of 0.1M sodium acetate and 3.4 ml 0.1M acetic acid were
> added to 27ml distilled water. Both solutions 1 and 2 were combined and 5ml
> of acetone was added. The slides were incubated for 60 min at room
> temperature, differentiated in 95% ethanol and dehydrated in several
> changes of absolute ethanol, followed by clearing in xylene with subsequent
> mounting (Kamino & Tarn 1991, Hillesheim et al 2011).
>
> An alternate method is to counterstain the immunohistochemical reaction
> with a methylene blue solution (method courtesy of Dr Vince Munro, St
> Vincents Hospital in Sydney):
>
> Staining Solution
> 2.38gm Sodium acetate
> 4.7ml Acetic acid
> 5gm Methylene Blue
> Make up to 1 litre with distilled water
>
> Procedure
> 1.  After immunostaining, wash slides in tap water
> 2.  Stain in Methylene Blue solution for 2 minutes
> 3.  Wash well in water
> 4.  Counterstain in Haematoxylin, wash well & blue as usual.
> 5.  Dehydrate, clear and mount
>
> Result: Melanin should stain green-blue.
>
> Hillesheim, P. B., Slone, S., Kelley, D., Malone, J., & Bahrami, S.
> (2011). An immunohistochemical comparison between MiTF and MART‐1 with
> Azure blue counterstaining in the setting of solar lentigo and melanoma in
> situ. Journal of cutaneous pathology, 38(7), 565-569
>
> Kamino H, Tarn ST (1991) Immunoperoxidase technique modified by
> counterstain with azure B as a diagnostic aid in evaluating heavily
> pigmented melanocytic neoplasms. Journal of cutaneous pathology,
> 18(6):436-439.
>
> Ravishankar, S., Nagarajan, P., Curry, J. L., Tetzlaff, M. T., Ivan, D.,
> Torres-Cabala, C. A., ... & Prieto, V. G. (2016). Giemsa is the optimal
> counterstain for immunohistochemical detection of BRAF V600E mutation
> status in pigmented melanomas. Journal of cutaneous pathology, 43(8),
> 722-724.
>
> Stefanović, D., Stefanović, M., & Nikin, Z. (2013). Romanowsky-Giemsa as a
> counterstain for immunohistochemistry: optimizing a traditional reagent.
> Biotechnic & Histochemistry, 88(6), 329-335.
>
>
> Regards
> Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
> Principal Scientist, the Children’s Hospital at Westmead
> Adjunct Fellow, School of Medicine, University of Western Sydney
> Tel: 612 9845 3306
> Fax: 612 9845 3318
> Pathology Department
> the children's hospital at westmead
> Cnr Hawkesbury Road and Hainsworth Street, Westmead
> Locked Bag 4001, Westmead NSW 2145, AUSTRALIA
>
>
>
>
> -Original Message-
> From: jayalakshmy p.s via Histonet [mailto:
> histonet@lists.utsouthwes

Re: [Histonet] Clarification about Immunohistochemistry

[jayalakshmy p.s via Histonet]

Thank you so much Susan and Tony. Let me prepare the reagents 
To Susan- can you please elaborate on the steps of the giemsa method.
To Tony Henwood - please tell what is meant by "bond washing"
Thanks
Dr. Jayalakshmy

On Tue, Apr 12, 2022, 9:58 AM Tony Henwood (SCHN) <
tony.henw...@health.nsw.gov.au> wrote:

> Hi there,
>
> I have had good results with Giemsa-like counterstaining (Stefanović  et
> al 2013, Ravishankar et al 2016)  :
>
> Azure blue, when substituted for hematoxylin as a counterstain in
> immunostain preparation, has been used to help differentiate melanocytes
> from melanophages. Azure blue preferentially stains cytoplasmic melanin
> granules blue-green, whereas melanocytes are highlighted by brown DAB
> chromogen. Melanophages, which contain melanin and lack melanocytic
> determinants, appear clear with blue-green granules in the cytoplasm
> (Hillesheim et al 2011).
>
> After the slides were bond washed for 4min and rinsed in distilled water,
> they were stained with a mixture of the following solution: 100 mg of Azure
> blue (Sigma) in 4 ml of distilled water. Solution 2 was prepared as
> follows: 0.6ml of 0.1M sodium acetate and 3.4 ml 0.1M acetic acid were
> added to 27ml distilled water. Both solutions 1 and 2 were combined and 5ml
> of acetone was added. The slides were incubated for 60 min at room
> temperature, differentiated in 95% ethanol and dehydrated in several
> changes of absolute ethanol, followed by clearing in xylene with subsequent
> mounting (Kamino & Tarn 1991, Hillesheim et al 2011).
>
> An alternate method is to counterstain the immunohistochemical reaction
> with a methylene blue solution (method courtesy of Dr Vince Munro, St
> Vincents Hospital in Sydney):
>
> Staining Solution
> 2.38gm Sodium acetate
> 4.7ml Acetic acid
> 5gm Methylene Blue
> Make up to 1 litre with distilled water
>
> Procedure
> 1.  After immunostaining, wash slides in tap water
> 2.  Stain in Methylene Blue solution for 2 minutes
> 3.  Wash well in water
> 4.  Counterstain in Haematoxylin, wash well & blue as usual.
> 5.  Dehydrate, clear and mount
>
> Result: Melanin should stain green-blue.
>
> Hillesheim, P. B., Slone, S., Kelley, D., Malone, J., & Bahrami, S.
> (2011). An immunohistochemical comparison between MiTF and MART‐1 with
> Azure blue counterstaining in the setting of solar lentigo and melanoma in
> situ. Journal of cutaneous pathology, 38(7), 565-569
>
> Kamino H, Tarn ST (1991) Immunoperoxidase technique modified by
> counterstain with azure B as a diagnostic aid in evaluating heavily
> pigmented melanocytic neoplasms. Journal of cutaneous pathology,
> 18(6):436-439.
>
> Ravishankar, S., Nagarajan, P., Curry, J. L., Tetzlaff, M. T., Ivan, D.,
> Torres-Cabala, C. A., ... & Prieto, V. G. (2016). Giemsa is the optimal
> counterstain for immunohistochemical detection of BRAF V600E mutation
> status in pigmented melanomas. Journal of cutaneous pathology, 43(8),
> 722-724.
>
> Stefanović, D., Stefanović, M., & Nikin, Z. (2013). Romanowsky-Giemsa as a
> counterstain for immunohistochemistry: optimizing a traditional reagent.
> Biotechnic & Histochemistry, 88(6), 329-335.
>
>
> Regards
> Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
> Principal Scientist, the Children’s Hospital at Westmead
> Adjunct Fellow, School of Medicine, University of Western Sydney
> Tel: 612 9845 3306
> Fax: 612 9845 3318
> Pathology Department
> the children's hospital at westmead
> Cnr Hawkesbury Road and Hainsworth Street, Westmead
> Locked Bag 4001, Westmead NSW 2145, AUSTRALIA
>
>
>
>
> -Original Message-
> From: jayalakshmy p.s via Histonet [mailto:
> histonet@lists.utsouthwestern.edu]
> Sent: Tuesday, 12 April 2022 1:59 PM
> To: histonet@lists.utsouthwestern.edu
> Subject: [Histonet] Clarification about Immunohistochemistry
>
> Hai all Histonetters
>  Please anybody clarify my this doubt if possible.
> When doing Immunohistochemistry for confirmation of Melanoma with DAB
> chromogen(brown color)the interpretation is not possible because of
> obscuring by the dense melanin pigment. We dont have any other color
> chromogen. I tried ihc after bleaching but the section gets detached even
> from charged slides. Is there any other effective way to do this?
> Thanks in advance
> Regards
> Dr. P S Jayalakshmy
> ___
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[Histonet] Clarification about Immunohistochemistry

[jayalakshmy p.s via Histonet]

Hai all Histonetters
 Please anybody clarify my this doubt if possible.
When doing Immunohistochemistry for confirmation of Melanoma with DAB
chromogen(brown color)the interpretation is not possible because of
obscuring by the dense melanin pigment. We dont have any other color
chromogen. I tried ihc after bleaching but the section gets detached even
from charged slides. Is there any other effective way to do this?
Thanks in advance
Regards
Dr. P S Jayalakshmy
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Re: [Histonet] Histonet Digest, Vol 220, Issue 8

[jayalakshmy p.s via Histonet]

Hello all, I would like to know whether Immunohistochemistry markers can be
used after its expiry date(atleast in resource poor countries). If yes, for
how much period of time and what is the criteria to look for?
Thanks and regards
Jayalakshmy

On Wed, Mar 9, 2022, 11:30 PM 
wrote:

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>1. Re: Post message to Histonet (Chenoa Hardwick)
>2. Re: formalin in OR (Greg Dobbin)
>3. posting (Chenoa Hardwick)
>4. Re: formalin in OR (John Garratt)
>
>
>
> -- Forwarded message --
> From: Chenoa Hardwick 
> To: histonet@lists.utsouthwestern.edu
> Cc:
> Bcc:
> Date: Tue, 8 Mar 2022 12:17:13 -0600
> Subject: Re: [Histonet] Post message to Histonet
> Please post to histonet:
>
> Job opening for histotechs (all shifts) and Pathology laboratory manager
> position (Utah)
>
> Contact che...@pathologywatch.com for inquiries.
>
> Thanks
>
> *Chenoa Hardwick*
>
> VP of Laboratory Services
>
> email: che...@pathologywatch.com 
>
> mobile: +1 972.351.0774
>
>
> 
>
>
> On Tue, Mar 8, 2022 at 12:08 PM Chenoa Hardwick  >
> wrote:
>
> > I would like to post a job opening on Histonet.
> >
> > Please see attached job description.
> >
> > Thanks
> >
> > *Chenoa Hardwick*
> >
> > VP of Laboratory Services
> >
> > email: che...@pathologywatch.com 
> >
> > mobile: +1 972.351.0774
> >
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> -- Forwarded message --
> From: Greg Dobbin 
> To: nancy.schm...@mercyhealth.com, histonet@lists.utsouthwestern.edu
> Cc:
> Bcc:
> Date: Tue, 8 Mar 2022 16:00:14 -0400
> Subject: Re: [Histonet] formalin in OR
> Hi Nancy,
> All routine specimens in our hospital are placed in formalin in the OR.
> Breast lumps and mastectomy specimens are sent up fresh so that they arrive
> STAT (to minimize cold ischemic times) and lymph nodes for lymphoma
> protocol are also sent up fresh. [*they actually send sentinel nodes up
> fresh too- it was just less confusing*]
>
> Diagnostic Imaging will place routine needle core biopsies directly in
> formalin (e.g needle cores of breast, prostate, non-lympoma lymph nodes and
> other misc. tissue masses). Lymph node cores for lymphoma protocol and
> renal cores are sent to the lab fresh, in a container on saline soaked
> Telfa pads.
>
> Endoscopy places all of their specimens directly in formalin. The derm
> clinic will place all routine skins directly into formalin but specimens
> destined for immunofluoresence are sent up to the lab fresh in a container
> on saline soaked Telfa pads. EBUS specimens are split between Cytology and
> Histology...the histo specimens go directly into formalin.
>
> Greg
>
> --
> *Greg Dobbin*
> 1205 Pleasant Grove Rd
> RR#2 York,
> PE  C0A 1P0
>
>
> *Everything in moderation...even moderation itself**!*
>
>
>
>
> -- Forwarded message --
> From: Chenoa Hardwick 
> To: histonet@lists.utsouthwestern.edu
> Cc:
> Bcc:
> Date: Tue, 8 Mar 2022 14:19:11 -0600
> Subject: [Histonet] posting
> Please post:
>
> Job open - Salt Lake City, UT - Pathology Lab Manager
> Relocation, sign on bonus available.
>
> Please contact Chenoa Hardwick at che...@pathologywatch.com 972-351-0774
>
> *Chenoa Hardwick*
>
> VP of Laboratory Services
>
> email: che...@pathologywatch.com 
>
> mobile: +1 972.351.0774
>
>
> 
>
>
> 
> Secured by Paubox - HITRUST CSF certified
> https://www.paubox.com
> 
>
>
>
> -- Forwarded message --
> From: John Garratt 
> To: Nancy Schmitt , "
> histonet@lists.utsouthwestern.edu" 
> Cc:
> Bcc:
> Date: Wed, 09 Mar 2022 01:28:25 +
> Subject: Re: [Histonet] formalin in OR
> I would not normally promote a product but I have seen the TissueSafe and
> SealSafe (Milestone) in use at a couple of hospitals for formalin
> management in the OR and in the lab and was most impressed.
>
> John
>
> Sent from ProtonMail for iOS
>
> On Tue, Mar 8, 2022 at 9:54 AM, Nancy Schmitt via Histonet <
> histonet@lists.utsouthwestern.edu> wrote:
>
> > Hello-
> > I would appreciate input on how you are getting formalin on to the
> specimens:
> >
> > * Is it done in OR
> > * Are specimens brough to pathology and add formalin there
> > * If so - does lab or OR add the formalin
> > * Other?
> > Are you Joint Commission?
> > Thank you!
> > Nancy Schmitt MLT, HT(ASCP)
> > Pathology Support 

[Histonet] Restain

[jayalakshmy p.s via Histonet]

Hello all,
How to restain effectively an old H stained faded slide.

Thanks
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