Question (55801) | BrdU Immunohistochemistry Kit (ab125306)

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Question

I have some questions about this BrdU kit.

Answer

To answer your questions about ab125306:
1) For suspension cells – is cytospin the only preparation method for suspension cells?
It is possible to prepare slides by preparing a 'slurry' suspension of cells and air drying them onto slides. Followed by dehydration fixing with 70 % etoh and processing. Cell morphology however is compromised for non adherent cells.
2) Other antigen retrieval methods may be used, should these be PIER as well or would HIER techniques be okay?
Proteases are required to reveal epitopes for the IHC BrdU kit.
3) Why can’t tissues/cells be rinsed after blocking step? If a customer accidentally does this, can they repeat just the blocking step and proceed with the protocol or do they need to repeat the denaturing step as well.
Rinsing after the blocking step may slightly increase background but shouldn't be 'terminal'. It is not required to repeat the denaturing step.
4) What is the purpose of the ethanol incubation step followed by xylene (this is after the counterstain) (mentioned on page 4)?
Ethanol {100 - 70% followed by PBS) is used after xylene to rehydrate the tissue. The procedure for dehydrating samples (prior to cover slipping) is the reverse. Incubate slides in 90% ethanol for 30 seconds, 100% ethanol for 30 seconds and xylene for 30 seconds (2 times each). Add 1-2 drops of mount- ing media and coverslip.

5) For paraffin sections, what are the guidelines for BrdU treatment of an animal [a) how much BrdU per animal; b) how long of a treatment with BrdU]?
Protocol attached.
6) For BrdU staining of cultured cells and cell suspensions: Under section A 3. Suitable slides are mentioned. Are any specific slides recommended? Do they need to be coated?
'Appropriate' slides refers to coated slides. Coated slides are required.
7) Should I want to perform an isotype control; what is the species, isotype and concentration of the detector ab?
It wil be difficult to use an appropriate isotype control - the concentrations of antibody and detector reagents in the kit are LOT specific and vary. A no BrdU control will provide a suitable control as all other reagents are used (except BrdU).
8) Will this product work with yeast?
The assay should work in any cell type that incorporates BrdU.
9) Can TBS be used in lieu of PBS?
TBS can be used however the assay has been optimized for use with PBS and we have no in house experience using TBS in the assay.

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