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9

As you may have realized, crystal violet can be replaced by a lot of dyes since the ethanol will wash out the dye from the gram negative cells. Methylene blue is a nice choice - malachite green may also work. A weak concentration of hydrochloric acid (3%) is also a possible replacement for the ethanol. A 3% HCl solution is used in the acid-fast stain but it ...


5

This answer addresses the question of how stains were developed historically, which may bring into perspective your overly modern assumptions. Most of these older (but stil very usefull!) staining techniques, such as haematoxylin (or rather haematein), cochenial/carmine etc. were inspired by the work of textile dyers of that era, starting somewhere in the ...


5

There is no specific dye for stem cells. You would have to do an immuno-histochemical staining for stem cell markers such as Sox2/Oct4 etc. Usually stem cells have a distinct morphology (round and clustered). You can use Leishman's (or Romanowsky-Giemsa) stain.


4

It depends on how much time elapsed between adding the concentrated antibodies to the diluting solution and adding the diluted antibodies to the slide. If it was just a couple of seconds, then I can abolutely see there being a difference. Even if it was longer - say 30-90 seconds, even - there potentially could be antibody gradients in the solution without ...


3

I'll amend this answer as I get details from you. What is your cytometer, and how are the optics set up? You typically have 1-5 lasers: UV (355nm), violet (405nm), blue (488nm), green/yellow (~561nm) and red (~633nm) lasers. Your FL number, FL1-4, typically refer to a particular PMT (photomultiplier tube) and filter combination. For example, a typical ...


3

All three methods could be used to measure the amount of DNA. However in practice, method 2 (estimation by dye brightness) typically works best in a normal workflow. It really depends on what you plan to do as your downstream application. Problems with method 1: Yield from gel purification methods is sometimes finicky and prone to loss. Absorbance of ...


3

You have several questions, please try to limit your posts to one question each. That being said, I'll try to address each of your concerns. Does the cold cause these cells to apoptose? No. The purpose of staining at 4°C and washing with cold buffer is twofold - to maintain viability (all cellular processes slow down at lower temps) and to prevent ...


2

The secretion pushes the cell organelles to the basal part. (although the fat cells have no polarity, their organelles have been pushed to one side of the cell: Comparing to adipose cells, the acinar cells secrete their specific proteins on one specific end of the cell, this is why in contrast with fat cells they have well defined polarity.


2

Yes, fixation of almost any kind can have effects on morphology. When you take a free flowing, protein spiked fat-blob (ie cell membrane), and make it rigid, you are going to get some differences. A fun visualization of this can be done by wrapping cellophane/shrink wrap around a serological pipette and dipping it in a dry ice and methanol bath. It's ...


1

This is described with better detail (and better prose) in John Baker's classic description at StainsFile, but to summarize: The procedure you're describing is a regressive hematoxylin stain with Harris hematoxylin. To start off with, let's bear in mind that hematoxylin, a compound from the wood of the logwood tree, is actually used as hematein, by ...


1

Disclaimer: Assumes you know how flow cytometry works. You've done a flow-based AnnexinV apoptosis assay here. Annexin A5 (AnnexinV) protein binds to phosphatidylserine (PS), which is typically in abundance of the inner leaflet of live cells. Upon the initiation of apoptosis, PS is known to flip to the outer leaflet. Thus, you can use AnnexinV conjugated ...


1

Taxonomic Classification: The most salient acid-fast bacteria are Mycobacterium spp., which are part of a phylum of gram-positive bacteria called Actinobacteria. This classification is based on 16S rRNA sequencing. However, based on genetic conservation, this paper makes the claim that they are more closely related to gram negative bacteria. Gram Reaction: ...


1

I think the issue here is the image is a 2D representation of a 3D object. It may well happen that the Barr bodies are below the Geimsa stained nucleus. This is the same reason we don't see peripheral nuclei in all muscle fascicles in a muscle cross section.


1

How about Brdu...? Normally, stem cells have active proliferation, their dividing are fast, thus they could uptake Brdu, and this is one of basic stem cell test in papers. I think it isn't too expensive, comparing with antibodies.... but you may need UV-light, and don't let children play Brdu. It is carcinogen, however, I think it only a very high dose can ...


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