1. Antibodies and immunofluorescence labeling
Green: primary antibodies against LAMP1 Protein (lysosomal associated membrane protein 1)
Antibodies and immunofluorescence labeling

2. A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation
What is an antibody?
Antibodies, or immunoglobulins, are glycoproteins composed of 2 heavy and 2 light chains.
Both light and heavy chains have different variable regions depending how the DNA is arranged and can form more than 15,000,000 combinations
“Somatic Recombination” is the mechanism by which some DNA is removed from the antibody coding region
Variable regions

3. How do you create a specific antibody?
A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation
How do you create a specific antibody?
Immunize mouse to desired antigen
Isolate B cells (mortal) from spleen
Cultivate myeloma cells (immortal)
Fuse both cell types
Separate individual cells and cultivate
Screen for antibody production
Multiply and harvest successful hybridoma cells

4. Different immunolabeling techniques
Immunofluorescence straining: antibodies are conjugated with a fluorescent dye that can be excited to emit detectable light
Immunoenzymological staining: antibodies are conjugated with an enzyme that catalyzes the transitions of an added substrate into an insoluble particle that can be localized
Immunocolloidal gold staining: antibody is conjugated with gold that is used as a marker

5. A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation
QUIZ 1!
Why can different antibodies bind to so many different biomolecules?
Why do you think the B cells are fused with myeloma cells?
What is the main difference between immunofluorescence staining, immunoenzymological staining, and immunocolloidal gold staining?

6. Function of immunofluorescence
Antibodies target specific biomolecules (antigens) and reveal their distribution and density throughout the sample via conjugated fluorophore
Green = AlexaFluor 488nm dye conjugated to actin; Blue = DAPI; Red = auto-fluorescence

7. Primary/Direct immunofluorescence
A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation
Primary/Direct immunofluorescence
Uses a single type of ‘antibody
The ‘primary’ antibody binds directly to target biomolecule (antigen) and fluoresces via the attached fluorophore

8. Secondary/Indirect immunofluorescence
A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation
Secondary/Indirect immunofluorescence
Uses 2 antibody types
The ‘primary’ antibody binds directly to the target biomelocule (antigen) but has no attached fluorophore
The ‘secondary’ antibody binds to the ‘primary’ antibody and fluoresces via the attached fluorophore

9. Example of primary/direct immunofluorescence
SLBP (stem-loop binding protein) antibody binding to hairpin loops on mRNA in Hep G2 cells (liver tissue with hepatocellular carcinoma)

10. Example of secondary/indirect immunofluorescence
Smooth muscle antibody binding to actin, vimentin, desmin, and tubulin (all microfilaments) in Hep G2 cells (liver tissue with hepatocellular carcinoma)

11. Primary versus Secondary immunofluorescence
A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation
Primary versus Secondary immunofluorescence
TIME
Primary:
Less steps require less time
Secondary:
Additional steps require additional time.

12. Primary versus Secondary immunofluorescence
Interatction with immunoglobulins
Primary versus Secondary immunofluorescence
COMPLEXITY
Primary:
Less steps in the protocol makes the experiment less complex
Secondary:
Additional steps, selection of secondary antibodies, and potential background interactions add additional complexity

13. Primary versus Secondary immunofluorescence
A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation
Primary versus Secondary immunofluorescence
COST
Primary:
Primary antibodies with an attached fluorophore (conjugated) are typically more expensive than unconjugated primary antibodies
Secondary:
Secondary antibodies are less expensive and can used against different primary antibodies

14. Primary versus Secondary immunofluorescence
A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation
Primary versus Secondary immunofluorescence
SENSITIVITY
Primary:
Less conjugated antibodies bind overall, so the signal is less intense
Secondary:
Several secondary antibodies bind to the primary antibody and produce an amplified signal

15. Primary versus Secondary immunofluorescence
A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation
Primary versus Secondary immunofluorescence
FLEXIBILITY
Primary:
Pre-conjugated primary antibodies have limited flexibility
Secondary:
Ability to use different secondary antibodies increase flexibility of experiment

16. Primary versus Secondary immunofluorescence
A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation
Primary versus Secondary immunofluorescence
SUMMARY!
Primary:
Less time required
Less complexity
Higher cost
Less sensitivity
Less flexibility
Secondary:
Higher time required
Higher complexity
Less cost
Higher sensitivity
Higher flexibility

17. QUIZ 2! Is primary or secondary immunofluorescence more complex?
A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation
QUIZ 2!
Is primary or secondary immunofluorescence more complex?
If you were attempting to detect a biomolecule that likely has very low concentration in the sample, would you choose to use secondary or primary immunofluorescence?

18. Immunofluorescence protocol for staining cell cultures
A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation
Immunofluorescence protocol for staining cell cultures
General Procedure
Coat coverslips in polyethyleneimine (for increased adhesion)
Rinse coverslips with water
Dry coverslips
Grow cells on glass coverslips
Rinse with Phosphate-based saline (PBS)

19. Immunofluorescence protocol for staining cell cultures
A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation
Immunofluorescence protocol for staining cell cultures
Fixation
Either Incubate cells with 100% methanol or 4% (para)formaldehyde in PBS
Wash cells three times with cold PBS

20. Immunofluorescence protocol for staining cell cultures
A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation
Immunofluorescence protocol for staining cell cultures
(Optional) Permeabilization
Incubate samples with PBS and % Triton X-100 (a detergent that improve antibody penetration)
Wash cells with PBS three times

21. Immunofluorescence protocol for staining cell cultures
A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation
Immunofluorescence protocol for staining cell cultures
Blocking and incubation
Incubate cells with
-1% BSA (bovine serum albumin)
-22.52mg/ml glycine (reacts with any excess formadehyde) in PBS + tween 20 (a surfactant) for 30 minutes
2. Incubate cells with primary antibodies in 1% BSA in PBS + tween 20 for 1 hour at room temperature

22. Immunofluorescence protocol for staining cell cultures
A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation
Immunofluorescence protocol for staining cell cultures
Blocking and incubation
3. Wash cells three times in PBS
4. Incubate cells with secondary antibody in 1% BSA for 1 hour at room temperature in the dark
5. Wash antibody solution three times with PBS in the dark

23. Immunofluorescence protocol for staining cell cultures
A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation
Immunofluorescence protocol for staining cell cultures
Mounting
Mount coverslip with a drop of mounting medium
Seal coverslip
Store in darkness

24. A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation
LAST QUIZ!
If you were examining a human cell culture with indirect immunofluorescence using bovine primary antibodies and rabbit secondary antibodies, what type of albumin serum would you use when blocking and incubating the cell culture?

25. A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation
General references

26. A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation
Academic References
1. Agrawal A, Godar DE. Simultaneous Detection and Semiquantification of DNA Damage in Normal and Apoptotic Cells: Triple-Immunofluorescent Labeling Using DAPI, Antibodies, and TUNEL. Applied Immunohistochemistry & Molecular Morphology. 2012;20:
2. Bordeaux J, Welsh AW, Agarwal S, et al. Antibody validation. BioTechniques. 2010;48:
3. Galluzzi L, Aaronson SA, Abrams J, et al. Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes. Cell Death Differ. 2009;16:
4. Zinchuk V, Zinchuk O, Okada T. Quantitative colocalization analysis of multicolor confocal immunofluorescence microscopy images: Pushing pixels to explore biological phenomena. Acta Histochem Cytochem. 2007;40:

27. Indirect fluorescence picture example
A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation
Indirect fluorescence picture example
Skin and vaginal tissues exposed to UV damage at time of damage (a,b), 1 hour post-damage (c,d), and 24 hours post damage (c,f)
DAPI = blue (nuclei)
TUNEL = green (apoptosis)
CPD (cyclobutane pyrimidine dimers) antibody = red (DNA damage)
Agrawal A, Godar DE. Simultaneous Detection and Semiquantification of DNA Damage in Normal and Apoptotic Cells: Triple-Immunofluorescent Labeling Using DAPI, Antibodies, and TUNEL. Applied Immunohistochemistry & Molecular Morphology. 2012;20: