Home › Cell Biology › Determination of Protein Expression Level in Cultured Cells by Immunocytochemistry on Paraffin-embedded Cell Blocks
Cell Biology JoVE (Open Access) Citable · DOI

Determination of Protein Expression Level in Cultured Cells by Immunocytochemistry on Paraffin-embedded Cell Blocks

DOI: 10.3791/57369-v
What you'll learn
  • ✓ Prepare cultured cell blocks for paraffin embedding and sectioning
  • ✓ Perform immunocytochemistry on paraffin-embedded cell sections
  • ✓ Quantify protein expression levels with morphological context
  • ✓ Compare immunocytochemistry to immunofluorescence methods
Protocol

Biopharma Insights Currently, immunofluorescent staining on fixed cells is the method of choice for determination of protein expression levels when morphological information is also necessary. This protocol presented herein provides for an alternative method of immunocytochemistry on paraffin-embedded cell blocks.

Difficulty
intermediate
Total time
~3–4 days (includes cell culture, fixation, embedding, processing, staining)
Model organism
HeLa cells
Biosafety
BSL-1

Steps

1
Serum-starve HeLa cells for synchronization

Culture HeLa cells and remove serum to synchronize the population and induce a defined physiological state prior to protein expression analysis.

▶ 00:40
2
Prepare cell blocks from cultured cells

Harvest and aggregate cultured cells into a cohesive block format suitable for downstream paraffin embedding and sectioning.

▶ 01:49
3
Process and embed cell blocks in paraffin

Dehydrate and infiltrate cell blocks with paraffin wax to create solid blocks that can be sectioned for microscopy.

▶ 03:01
4
Perform immunocytochemical staining protocol

Execute antigen detection and antibody labeling on paraffin-embedded cell sections to visualize target protein expression.

▶ 03:50
5
Analyze histological and immunocytochemical results

Examine stained sections microscopically and quantify protein expression levels in relation to cellular morphology.

▶ 06:50
💬 Comments coming soon