How to do imunostaining?

More Adity Pore's questions See All

How to prevent drying in 4% PFA fixed slides? What are some best techniques to store fixed slides for several weeks/months?

I am currently storing my fixed slides at 4 C. These slides tend to dry out. Are these slides still good to use? can these slides be revived by adding liquid? I have attached some images .

22 June 2020 7,070 5 View

What would be a suitable EMT marker for breast CTCs?

There are several EMT markers in cancer, such as vimentin, n-cadherin, SNAIL, TWIST, ZEB etc. How do you decide which one to use?

13 June 2020 8,901 2 View

What are some drawbacks of conjugated antibodies? Why are they not used as popularly as indirect immunofluorescence techniques?

I have been using conjugated antibodies for staining cancer cells. And I try preserving the fluorescence signals using Prolong antifade with Dapi. However, after 24-48 hours, the only signal I see...

09 June 2020 4,679 1 View

What is the difference betweeen mesenchymal markers and stem cell markers in CTCs?

For example CD44 is a stem cell marker, whereas Vimentin is an EMT marker. Mesenchymal cells are called MSCs (Mesenchymal Stem Cells), then why are there different markers for stem cells and...

01 June 2020 2,637 5 View

How long can mammalian cancer cell lines be stored in -80C?

I wanted to know if we can store frozen cells in -80 C refrigerator. These cells are stored in liquid nitrogen tank (-200 C) as of now. But as liquid nitrogen vaporizes, we need to keep refilling...

23 March 2020 8,753 10 View

How long does it take for PDMS to lose hydrophilicity after plasma treatment?

I want to make a hydrophobic microfluidic device, I plasma bonded the device on a PDMS slab. Since I used plasma treatment, I wanted to know how long will it take for PDMS to lose hydrophilicity.

05 February 2020 9,387 8 View

No title

My cells are fixed on a coverslip and are stained with DAPI, Cytokeratin and CD45. What steps should I take after staining the cells?

18 September 2019 1,417 4 View

Do cells stick to the substrate after you put 4% PFA?

If I have cells on a glass slide and I put 4% PFA on it, are the cells going to be stuck on that particular position always? Will the cells move if I add PBS to it? What exactly does fixation do...

18 March 2019 5,673 3 View

What is the small dust like particulate matter floating in cell culture media?

I saw small dust like particles floating in my cell culture media (DMEM) and in the cell culture flask as well. My cells are growing fine in this media. Also, I see the same particulate matter in...

07 June 2018 3,161 7 View

Why does intensity of an image containing fluorescence increases, with increasing dilution?

Co is the initial concentration of the dye, while Cn is the concentration of the dye after dilution. Intensity of the images are found using ImageJ as well as MATLAB, there is hardly any...

19 June 2017 3,119 3 View

Why does blocking with my immunogenic peptide cause nuclear fluorescence in ICC?

Hi all, I am validating a polyclonal antibody raised against human ANT4, through WB and ICC. In most of my controls, the antibody shows very little off-target binding, but when I pre-incubate the...

03 March 2021 5,999 3 View

Have someone successfully used the ER alpha (Santa Cruz sc-8002) antibody in immunostaining ?

Santa Cruz now recommends this new antibody (F-10) but I can't find a publication using this reference. I'm working on human et non human primate hypothalamus and so far, it doesn't work. Is...

02 March 2021 10,003 2 View

Does Napabucasin soluble in dichloromethane and in ethanol?

I need to prepare a solution of Napabucasin (inhibitor of cancer cell stemness) in dichloromethane. The drug is expensive, so maybe somebody already tried to do it. If it is soluble, what is the...

02 March 2021 9,945 3 View

Help request in CST microwave?

im working now on TPV solar cells and metamaterials and i realy need our help in CST microwave i have a tpv cell simulation and i want to extract particullarly the absorbtion of only the active...

02 March 2021 2,246 2 View

Could you please recommend some good journal that accept very long revie papers (approximately 6000+ words) in neurosurgery, cancer biology ?

01 March 2021 8,087 3 View

Which cell line should we use to perform biological dosimetry experiments?

We are preparing some experiments based on irradiating cells under different conditions in order to evaluate the effects in terms of DNA damage, genetic expression, etc. As our project is...

01 March 2021 3,355 3 View

Is CD44 degraded in lysosome together with hyaluronan after CD44 mediated endocytosis of hyaluronic acid?

Also when RHAMM binds hyaluronic acid, they get internalized, will RHAMM also be degraded? Or both CD44 and RHAMM will be transferred back to the cell membrane? Asking for breast cancer cell line...

01 March 2021 8,169 2 View

Dapi fluorescence protocol for colon cell lines ?

28 February 2021 2,534 2 View

Does RNAiMAX transfection reagent work in serum free medium?

I diluted siRNA and RNAiMAX in opti-MEM and added to the cells which they were in the growth medium. Is it a right way? or should I culture cells in the opti-MEM medium for a while and not in...

26 February 2021 10,041 3 View

Has anyone had luck using cell lines other than HEK 293 (such as COS1) to package lentivirus?

I am looking to package lentivirus in endothelial cells but I am opposed to using 293 cells due to personal ethics regarding the source. I have done some reading and saw that COS1, COS7, & CHO...

25 February 2021 3,030 1 View

Saleh Alkarim

Hello

Kindly look at link and pdf here

Article MIB-1 immunostaining on cytological samples: A protocol with...

Good Luck

Srinivas Pittala

Hi,

To do immunostaining for CELLS, we use immunocytochemistry(ICC) and/or immunoblot(western blot).

Regarding ICC, you can see this document https://images.novusbio.com/design/BR_ICCguide.pdf

http://www.ihcworld.com/protocol_database.htm#Histo-IHC

Primary antibody selection is depends on your research objective, protein of interest. After selecting the protein to see in cells, for example EGFR. You can search for antibody against EGFR from different companies. Depending on your budget stick to one company like abcam or CST, then select the antibody that can react with HUMAN cells as your MCF-7 cell line derived from human, go over the datasheet of that antibody carefully about the antibody applicable to use in WB/ICC/IHC/IF, check cross reactivity, concentration/dilution to use and ant previous user comments about it.

secondary antibody should be against from primary ab host species. if primary ab raised from rabbit, secondary should be mouse/goat/donkey raised against rabbit

Good luck

Han Zhang

Hi Adity, you can search a standard protocol for Western blot online, or you can ask for a protocol from other labs. Western blot is the most common way to detect protein levels. As for the antibodies, firstly, you need to decide which protein you are interested in, then you search for a specific primary antibody for this protein; secondly, you choose a secondary antibody based on the primary antibody due to the animal class. For instance, if the primary antibody is a rabbit one, you need to buy an anti-rabbit secondary antibody, if it is from mouse, you need an anti-mouse secondary antibody.

Hope it is helpful for you!