What is this that I am seeing during cryosectioning?

If A human body of 75 Kg. and 50 Kv X-ray tube and 10 cm in contact and current I=10mA. Calculate Dose Rate which in (Gy/s) = (mA * Exposure Time * Conversion Factor) / (Distance^2) Where: mA (milliamperes) = 10 Exposure Time (seconds) = 10 & for X-rays at 50 kV = 0.175 Gy/(s*mA). ?

Hello scientific community on Research Gate,

It has been a rough week as I have been troubleshooting to resolve mediocre paraffin sections.

Initially, I thought it was due to improper fixation (inadequate 10% NBF recipe) of my specimen. Then, I was made aware that absolute EtOH is not available in our lab and we were using 96.6% EtOH as a substitute. Therefore, it was sensible to assume that incomplete tissue dehydration could account for the observed artifacts on the paraffin ribbon. I managed to find some 99.9% EtOH and repeated the procedure, but to no avail.

It seems that the microtome blade, that I replaced, is cutting rather around (and not through) the tissue which leaves a perforation on the ribbon corresponding to my embedded specimen. On better instances, I would manage to obtain a (semblance of) section of my specimen with noticeable lacerations and tears.

My hunch is that the paraffin has deteriorated (expired since 2008) and is poorly impregnating the specimen.

As per your anecdata, what is causing this horrendous (at best) paraffin section?

Is this artifact most likely attributed to processing (i.e., fixation, dehydration, clearing, infiltration) or defective material?

To guide your diagnostic input, here's an approximate description of the protocol:

Fixation

10% NBF - 48h

Dehydration

60% EtOH - 1hr

70% EtOH - 1hr

80% EtOH - 1hr

90% EtOH - 1hr

100% EtOH - 1hr

(All of which are calculated/prepared from a 96.6% EtOH bottle deemed as 100%)

Do you think I am being overzealous to procure 100% ethanol or the available 96.6%EtOH would suffice for dehydration?

Clearing

Xylene - 1hr

Wax infiltration

In paraffin (Overnight, oven @70°C).

P.S. I have included snapshots (1, 2, 3 — from bad to worst) of the modest chef-d'oeuvres that I've produced for the past 2 weeks and it's borderline frustrating.

Your compassion and expertise are greatly appreciated.

Thank you!

Hi all,

Trying to section e14.5 mouse cortex and keep getting these tears along the lateral ventricles. I'm trying to look at neural progenitor populations along the lateral ventricles so tearing there is no good. Brains were fixed in 4% PFA overnight then put in 30% sucrose for ~72 hrs until they sunk. Tissue was embedded in OCT and put in dry ice for ~20 minutes to freeze. Sections were cut at 8um in a cryostat where chamber temp was -20C and stage temp was -17C. Any tips on how to avoid these tears would be much appreciated.

I recently ran a Western blot, and some of the bands appeared to be partially missing, as if they were cut in half. I looked online for possible solutions, but most troubleshooting guides do not address this issue. What could have gone wrong?

We've been trying to transduce THP-1 cells with some P2X7 constructs (Blasticidin resistance gene also present) using Lentivirus and Blasticidin selection. This has worked well in HEK293T cells before but doesn't seem to be working in THP-1s as after Blasticidin addition all the cells die within a brief period. Not just that, the GFP control transduction doesn't seem to have worked either.

Some ideas I had was perhaps the Blasticidin concentration is too high or we add it too fast, killing too many cells at once, causing the remaining live cells to die as well? Since the virus has previously been tested and had worked in HEK293T cells I doubt it is that, but we have also made new viral stocks.

I used muscle cells as sample, and the western blot band appear grey and blurry. I used non-fat milk for blocking, does this has any effect? This is my 10th time trying and the band still appear grey and blurry. I've done the protocol correctly, is there any other things I should consider doing or are there hacks for this problem? Thank you. Any advices will help a lot and appreciated.

I am new in the radiotherapy field and I need to set some experiments to determine the sensitivity of several cells lines to radiation.

I read a lot of papers and they look like to achieve my goal They use a technique called survival fraction assay, which is simply a clonogenic assay with some modifications.

My questions are:

How long take a cell line to die after radiation?

Why I can not use, for example MTT assays to check viability 72 h after radiation (different doses) and determine the IC50?

I am seeding cells and irradiating them with different doses and then doing MTT 72 h later. But my cells even at the highest doses of radiation, looks like controls. Do you know why??? They need more time to died o what?

I appreciate your helps in this matter.

Best

As described in the title, we usually perform puromycin selection because the plasmid contain such resistant gene, still some of our lab member does not change medium for a period of time until the observation of cell growth, while others replace puromycin-containing medium everyday. Therefore, does anyone know if there's information regarding half-life of puromycin in cell culture medium to optimize cell selection?

I am preparing for a proliferation assay where I have infected KP230 cells with shScr, shCol18a1#35, and shCol18a1#37 viruses. These shRNA viruses are made with the pLKO.1 vector and have puromycin resistance. I am at the stage of antibiotic selection, 48 hours after infection, where I will use 0.2 ug/mL puromycin for each of my three samples. Overall, the three 6 cm plates will have 18,000 KP230 cells, 0.6 uL puromycin, and 3 mL 10% growth media. I am advised to wait another 48 hours after antibiotic selection before typsinizing and seeding for the proliferation assay (growth curve).

Why does antibiotic selection take 48 hours? My protocol applies to other cell lines too such as HT-1080, STS-109, and STS-148 cells, so is 48 hours a standard among most cell types? What about using a different antibiotic on other resistant genes? Can anyone explain biologically what happens during these 48 hours with time stamps? What happens if I only give it 24 hours? I appreciate all your responses in advance.