Genomics and Molecular Diagnostics

Immunocytochemistry and Immunofluorescence

Name

: Bryan Liew Yoong Jie

Student ID

: 00000014495

Course

: BM 1/14

Group

: Group 4

Introduction
Immunofluorescence has always been used in clinical diagnostics and in the field of
research as it is one of the most powerful and versatile technique to study proteins in a cell by
fluorescent staining the proteins and then observing it under fluorescence microscopy.
Immunofluorescence technique uses both fixed and fresh samples. Antibodies would be
conjugated chemically to fluorescent dyes such as tetramethyl rhodamine isothiocyanate
(TRITC) or fluorescein isothiocyanate (FITC). These labelled antibodies would then bind to a
specific antigen which is then detected through fluorescence techniques.
Direct and indirect immunofluorescence are the two main methods in
immunofluorescent labelling. Direct immunofluorescent is done by chemically conjugating
the antibody against the molecule of interest to a fluorescent dye. For indirect
immunofluorescent, the primary antibody which specific tot eh molecule of interest is
unlabelled and so a secondary anti-immunoglubulin antibody which had been tagged with a
fluorescent dye would be directed to the constant region of the primary antibody. So, for this
particular experiment, the method used to detect the MCF-7 cells is indirect
immunofluorescent where the primary antibody used is cytokeratin 8 and the secondary
antibody used is goat polyclonal to mouse IgG.
Programmed cell death is called cell apoptosis. Death receptor-stimulated extrinsic
signalling or mitochondria-related intrinsic signalling actively regulates cell death and so, this
would induce a cascade of caspase-mediated photolytic degradation of substrates. This results
in the cells undergoing a series of biochemical and morphological changes and so this
provides the basis for a number of methods in detecting apoptotic cell. So, the objective of
this experiment is to perform immunofluorescence study on the human breast cancer cells by
using 4,6-diamidine-2-phenylindole dihydrochloride (DAPI) staining the nucleus blue and a
secondary antibody which has been tagged with FITC staining the cytokeratin 8 green.
Materials
MCF-7 cells, Phosphate buffer saline (PBS), BSA, Tween-20, PBST(0.1% Tween 20 in PBS),
Methanol, Acetone, DAPI, Primary antibody(Cytokeratin 8), Secondary antibody(Goat
polyclonal to Mouse IgG- H&L (FITC))
Methods
Staining of MCF-7 cells
The medium in the tissue culture plate was first removed and 2ml of phosphate-buffered
saline (PBS) was used to rinse the tissue culture briefly. 2ml of ice cold methanol:acetone
(1:1) was added in and was incubated at -20C. Before air drying the plate, the cold
methanol:acetone was removed first. The sample was washed twice with 2ml of ice cold PBS and
later incubated with 2ml of 1% BSA in PBST for 30 minutes. The 1% BSA/PBST solution was then
removed and the cells were incubated with 1ml of the diluted primary antibody 1:100 in 1% BSA in
PBST for 30 minutes at room temperature. The solution was decanted and the cells were washed with
2ml of PBS, for 5 minutes each. Cells were then incubated with 1ml of the secondary antibody
conjugated to a fluorochrome(1:4000) in 1% BSA in PBST for 30 minutes at room temperature. The
secondary antibody solution was decanted and washed three times with 1ml of PBS for 5 minutes
each in the dark. Cells were then rinsed with 2ml of PBS.

Cytokeratin 8 detection by immunofluorescence

The image was observed and captured under an inverted fluorescence microscope using
multiple magnifications. The image was saved and is pasted in the report below. The cells
were then stored in the dark at -20 C or 4 C if further analysis is needed.
Results
Comparing MCF-7 cells in bright light and fluorescence
To evaluate the difference between the image captured using bright light and fluorescence
microscope. Where the MCF-7 cells had been stained with DAPI and FITC. Figure one
shows the MCF-7 cells under bright light microscope while figure 2 and 3 under fluorescence
microscope.

Figure 1: bright light microscopy of MCF-7 in PBS

Figure 2

: MCF-7 cells viewed under fluorescence microscope nucleus of cell stained

blue by DAPI

Figure 3

:MCF-7 cells viewed under fluorescence microscope showing merge image of

nucleus stained in blue and cytokeratin 8 stained green

Discussion

References
http://www.breast-cancer-research.com/content/pdf/bcr2889.pdf
http://www.dako.com/08002_03aug09_ihc_guidebook_5th_edition_chapter_10.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1929551/pdf/pubhealthreporig001180078.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4314999/