[Histonet] Sample retention policy and tracking

2021-06-08 Thread Igor via Histonet 
Hello fellow histonetters,

I wanted to pick your minds with the following 2 questions pertaining to
storing and trackingsamples in the research environment :

1. How do you track samples that you store long term? As far as slides and
cassettes go, it makes sense that a lot of people use barcodes.  Does
anyone also store and track cell lines? How do you track them?

2. Do you have a sample retention policy for slides, cassettes, clinical
samples and for cell lines?? If you do not mind sharing some details on how
long you keep each kind of sample,  I would greatly appreciate it!

Thank you in advance!

Igor Deyneko
Scientific Operations Manager
Novartis Institutes for Biomedical Research
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2021-06-08 Thread Pam Barker via Histonet 
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Re: [Histonet] Problems with DIF staining of C4d

2021-06-08 Thread Morken, Timothy via Histonet 
Beth, which C4d are you using? And it is a FITC-labeled primary?



We use it as a two-step rather than FITC-labeled primary. That increases the 
sensitivity significantly. Gloms have C4d so act as an internal control.



We use
C4d antibody, 100ul vial, unlabeled primary.  Diluted 1:200 in Dako or Bond 
diluent.
Quidel, Cat#  A-213

Secondary Ab:
Antibody, Goat anti Mouse IgG H+L FITC 1.5mg. Diluted 1:120 in Dako or Bond 
diluent.
Jackson Immuno Research
115-095-062


We store the concentrate in the fridge. We use it daily so go through it pretty 
quickly, but we have it validated for two years at 2-8C. We make fresh 
dilutions a couple times per week.



We stain on the Bond Autostainer, but manual will work fine as well.





Tim Morken

Supervisor, Electron Microscopy/Neuromuscular Special Studies

Department of Pathology

UC San Francisco Medical Center



-Original Message-
From: O'Neil, Beth A. via Histonet 
Sent: Tuesday, June 08, 2021 10:32 AM
To: Histonet 
Subject: [Histonet] Problems with DIF staining of C4d



We perform manual DIF staining at our facility and C4d has always been a 
problem.  We end up repeating the stain more times than not due to lack of 
staining.  We aliquot the FITC and freeze at -70C until ready for use.  We also 
started doing the same with the C4d.  Sometimes it works and sometimes it 
doesn't.  My pathologist said it is not the FITC but the C4d itself.  I have 
contacted the manufacturer of my C4d but they are very slow to respond.  Any 
suggestions or experiences would be appreciated.



Beth ONeil

WVU Medicine, JW Ruby Memorial Hospital



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[Histonet] Problems with DIF staining of C4d

2021-06-08 Thread O'Neil, Beth A. via Histonet 
We perform manual DIF staining at our facility and C4d has always been a 
problem.  We end up repeating the stain more times than not due to lack of 
staining.  We aliquot the FITC and freeze at -70C until ready for use.  We also 
started doing the same with the C4d.  Sometimes it works and sometimes it 
doesn't.  My pathologist said it is not the FITC but the C4d itself.  I have 
contacted the manufacturer of my C4d but they are very slow to respond.  Any 
suggestions or experiences would be appreciated.

Beth ONeil
WVU Medicine, JW Ruby Memorial Hospital

Confidentiality Notice: This e-mail message, including any attachments, is for 
the sole use of the intended recipient(s) and may contain confidential and 
privileged information. Any unauthorized review, use, disclosure or 
distribution is prohibited. If you are not the intended recipient, please 
contact the sender by reply e-mail and destroy all copies of the original 
message.
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[Histonet] Issue with Technovit 7100 - nerve samples

2021-06-08 Thread Chaitanya Kolluru via Histonet 
Hi,

I'm working on a protocol to embed human nerve samples (about 3 mm in
diameter, 5-7 mm in length) in glycol methacrylate and cut 2-3 micron thick
sections.

The sectioning seems fine at the start, but as I section deeper into the
sample, I find a lot of gaps forming in between the fascicles, which I'm
assuming are air bubbles.

I'm attaching an image of the block surface after sectioning about 500
microns into the sample. I'm also attaching a text file of my protocol if
that can help troubleshoot the issue.

Any help on how I might be able to reduce this artifact would be much
appreciated. If an alternative resin would alleviate this issue, please do
let me know.

Thanks in advance,
Chaitanya

Link to image of the block surface:
https://cwru.box.com/s/5dsd7ngazlhf0jxga4apqh47a75uzb84
1. Samples were extracted from cadavers and left in formalin for more than a 
couple of month.
2. Cut 5 mm long nerve segments and rinse in PBS for 1h. Post-fix nerve in 2% 
osmium tetroxide for 2 hours.
3. Perform gradual dehydration in increasing steps of ethanol, about 2 steps 
per day for a total of 3 days (with rotation).
4. Pre-infiltrate specimen with a 1:1 mixture of 96% ethanol and 100% GMA 
solution for 2 days. Perform vacuum cycles for about 30 min everyday with 35 
psi in a vacuum pump.
5. Immerse sample in the infiltration mix (1g Hardener 1 in 100 ml GMA) for a 
total of 6 days, with 2 changes of fresh infiltration mix in between. Same 
vacuuming protocol as previous.
6. Polymerize with 15 parts infiltration mix and 1 part Hardener 2.
7. Polymerize in Teflon molds in vacuum at room temperature (25 deg C) for a 
couple of hours, followed by polymerization in the oven at 37 deg C. I see that 
the blocks are usually soft after this, so I let the samples continue to sit in 
the mold in the oven at 55 deg C for 48 hours.
8. Remove from mold with Technovit 3040, clamp on the motorized microtome 
(Microm) and section with tungsten carbide knife at 13 degrees clearance angle 
with a slow speed setting at 2-5 micron section thickness.___
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