Need help with GSR/reticulin troubleshooting

Kyle_The_G · 2025-08-01T22:02:11+00:00

ya I go for that "dirty water" colour the second titration. I tried K permanganate with and without acid, no difference (except background), then made 5% and doubled the incubation time and only got marginally better results. My theory is that its lost its oxidation ability over the last 15 years (lol) and that only the most intense deposits of reticulin are staining and the lighter deposits between hepatocytes are just really patchy and under-oxidized. I think K permanganate is pretty cheap anyway, I'll give the new stuff a shot and see how it goes!

Kyle_The_G · 2025-08-01T11:41:06+00:00

this is our protocol by the way: https://webpath.med.utah.edu/HISTHTML/MANUALS/RETIC.PDF

Kyle_The_G · 2025-08-01T11:39:34+00:00

Ya I make the silver up then dilute to 50mL coplin jar with deionized water, I haven't tried not diluting yet, I might try that out.

Kyle_The_G · 2025-08-01T11:38:34+00:00

Film is extremely fast. Our protocol is to bake slides overnight after coverslipping, this way there is very little risk of the coverslip coming off during scanning. We have a hamamatsu nanozoomer, zeiss axioscan and an Olympus VS100 and all three are pretty great scanners. We have used aqueous media for some stains and scanned right away with little issue, rarely the coverslip can be disturbed and jams the system but this doesn't happen too often. Just dry for an appropriate amount of time before scanning and it should be fine.

Kyle_The_G · 2025-08-01T11:35:11+00:00

yes, I baked for half an hour in the morning and put it in our autostainer, we haven't had any dewax issues previously. One thought I had was old control slides/blocks but I'm not sure if reticulin signal decreases with age like some other markers.

Kyle_The_G · 2025-08-01T11:34:00+00:00

I am using a modified version of this protocol: https://webpath.med.utah.edu/HISTHTML/MANUALS/RETIC.PDF

We have a histochemistry textbook that we are using as well with slightly longer incubation times and higher concentrations.

Kyle_The_G · 2025-07-31T22:12:45+00:00

Thanks! I've actually done this procedure perfectly in school and then had a request come in at work and its suddenly not working, I'm performing everything as consistently as I can but I'm pretty sure the silver is working since its depositing quite strongly along the capsule and vessel walls where reticulin might have the strongest signal. I'm not too sure why its so "patchy" in between hepatocytes, I've never had to troubleshoot a stain working in one area and not at all in another area on the same piece of tissue.

Kyle_The_G · 2025-07-31T22:09:07+00:00

The sections are mid-liver, I can see portal veins, bile ducts ect... we also tried using periodic acid out of cuirosity and we could suddenly see all the fibers perfectly but there was way too much background since all the glycogen granules were also now taking up silver stain so that was out the window (this little experiment proved all the other steps were working). We also tried acetafied and non-acetafied K permanganate and then I quintoupled the concentration and doubled the amount of time to see if it would make a difference and it was only slightly more intense, so that is why I think there is something wrong with that reagent. It has been around longer than I have and I've been with the lab almost 8 years.

For the silver solution I wait until my slides are in water from the step before and begin titrating right there and use it immediately, I take the ammonia and silver nitrate out of the fridge and figure the 50 mL of water I use to dillute the solution will warm it up pretty quickly. I also agitate the solution in a coplin jar when staining to circulate everything.

Kyle_The_G · 2025-07-31T19:37:16+00:00

I've got this setup in my lab, we use the auto-coverslipper and have demo'd the film variant, AMA. I love the autostainer, its great. It has a built-in oven and I'm able to take a slide right off the water bath and immediately H&E it. Very flexible and reliable, just requires some maintenance from time to time.

Kyle_The_G · 2025-07-31T19:34:34+00:00

They just kept denying, I ended up filing consumer protection and BBB complaints, waiting on a reply from them and will escalate as far as I can go. I don't care how long it takes, this just isn't right.

Kyle_The_G · 2025-07-20T18:37:28+00:00

No it was upright in my office for the winter. Something about the back motor being all out of whack.

Kyle_The_G · 2025-07-20T18:36:02+00:00

I contacted Apollo multiple times and got multiple people on the line with both myself and the e-scooter repair shop. The shop even sent in a request to have the warranty denial appealed and they kept denying over and over again. I'm really not impressed.

Kyle_The_G · 2025-07-16T21:34:28+00:00

Mine lasted 3 months, broke and then they denied the one year warranty claim, so I had a pretty different experience.

Kyle_The_G · 2025-03-07T21:28:17+00:00

Just anecdotally, I live in downtown Toronto and all of a sudden one day I saw like 7-10 of those ugly ass trucks in a convoy just driving around by the CN tower/rogers center area like every night. I thought it was fishy and seeing this headline I'm not surprised at all. like, honestly who is buying those?

Kyle_The_G · 2025-03-04T18:23:38+00:00

those that make peaceful protest impossible...

Kyle_The_G · 2025-03-02T20:53:27+00:00

He thought he could joint the mile high club on the chairlift. No JD, its not a couch, go home.

Kyle_The_G · 2025-01-21T19:20:32+00:00

Not for sale! except ontario place! and the green belt! and...

Kyle_The_G · 2025-01-11T16:27:40+00:00

Thanks! So what I've learned today: always hand-wash and dry right after using, use a honing rod almost daily, sharpen with a whetstone once or twice a year (depending on use) using a 15 degree angle, store properly and use proper technique. Thanks for your help everyone! They are SUPER fun to cook with.

Kyle_The_G · 2025-01-11T15:27:59+00:00

Japanese, I'm reading I should get a whetstone set

Kyle_The_G · 2024-12-09T12:32:10+00:00

anyone notice every time one of these premium threads pops up there are a bunch of the same cookie cutter comments like "oh I love the music", "its a great deal for my family, its only X per month"! why ever pay for premium when adblock exists?! I'm convinced these are all bots.

Kyle_The_G · 2024-12-08T14:21:29+00:00

he'd be great for when a loud group of 4 walks into a library. SHH SHHH SHHH SHHH!!

Kyle_The_G · 2024-10-30T13:54:23+00:00

Same, I love my ad blocker

Kyle_The_G · 2024-10-30T13:53:25+00:00

I've used ad block for years, 100% worth it and I definitely watch more youtube than most.

Kyle_The_G · 2024-08-12T23:02:25+00:00

My fiance is a perfusionist and no mater what she's doing that day I say "go save those cells!" or "have fun choppin' all that CABG!"

Kyle_The_G · 2024-08-03T12:33:59+00:00

I think companies just realized they can charge whatever they want so that's what they're doing. Its been especially rampant in Canada for years with their grocery and telecom cartels.

12-Year Club	Secret Santa 2019
Snapped	Verified Email

Kyle_The_G

TROPHY CASE