How do you Immunostain suspension cells?

Can someone help me with Immunostaining of suspension cells?

  1. Treat the cells with MMC.
  2. Release and collect the cells.
  3. Wash the cells with PBS – centrifugation.
  4. Fix the cells in PFA – 10 min – RT.
  5. Wash the cells with PBS – centrifugation.
  6. Permeabilize the cells in 0.1% TritonX in PBS – 15 min – RT.

What is immunostaining used for?

Immunostaining is used in cell biology to study differential protein expression, localization and distribution at the tissue, cellular, and subcellular level.

How do you prepare cells for immunostaining?

Cell preparation for suspension cells

  1. Centrifuge the cell suspension at 1,500 rpm for 5 min, discard supernatant.
  2. Wash cells with 1 mL of 1X PBS, and obtain a pellet by centrifugation at 1,500 rpm for 5 min.

How do we adhere cells to a slide?

Poly-lysine coating protocol: Nearly all types of adherent cells will adhere to a poly-lysine coating, making it the most popular coating choice. To coat your slides with poly-lysine, add enough 1:10 poly-lysine solution* to cover the tops of each of your cover slips.

How do you fix a suspension cell?


  1. Prepare a fresh solution of 4% paraformaldehyde in PBS.
  2. Wash the suspension cells twice with PBS by centrifugation at 200g for 5 minutes.
  3. Carefully resuspend the cell pellet in 4% paraformaldehyde in PBS at ~106 cells/ml.
  4. Wash the cells by centrifugation at 200g for 5 minutes and resuspend in PBS.

How do you mount suspension cells?

A simple method for detecting intracellular antigens in cells that grow in suspension is to attach the cells to a solid substrate before fixation. This can be achieved by the use of a cytocentrifuge. For surface staining, suspension cells can be attached to slides by cross-linking with poly-l-lysine.

What is a drawback of immunocytochemistry?

The disadvantages of IHC are as follows: IHC stains are not standardised worldwide. While the cost of the procedure is relatively inexpensive, the equipment needed to perform IHC is costly. Quantifying results is difficult. IHC is subject to human error.

What are immunohistochemical markers?

Immunohistochemical tumor markers are proteins that help doctors tell the difference between different types of cancer. Mesothelioma-related proteins such as calretinin, WT-1 and podoplanin help pathologists differentiate mesothelioma from other cancers such as lung cancer.

How do you fix if cells?

All incubation steps take place at room temperature.

  1. Wash the cells twice and use tweezers to carefully place the coverslip with upturned cells into the humidified chamber.
  2. Fix with 4 % formaldehyde for 10 minutes and wash 3 ×.
  3. Permeabilize with 0.1 % TX-100/PBS for 15–20 minutes and wash 3 ×.

How do you fix suspension cells?

How do you fix a suspension cell in a microscope?

To fix by cross-linking, add an equal amount of 4% paraformaldehyde to your 2 x 106 cell/ml suspension to create a 1 x 106 cell/ml suspension. Then incubate your cells in this solution for 10 minutes at room temperature.

Can you cytospin fixed cells?

cytospin is to let cells on a coverslip or slide to be stained or labeled… If cytospin after fixation is not possible: You can do IF-taining in tube (1. Antibody, washing steps 2.