The document provides instructions for using immunofluorescence techniques to detect antibodies. It describes how to prepare cell samples and reagents, and outlines two main methods - indirect immunofluorescence and cell membrane fluorescence staining. Indirect immunofluorescence involves incubating cell samples with antibody samples to be tested, then a fluorescent secondary antibody, and viewing under a microscope. Cell membrane fluorescence staining uses living cell suspensions incubated at 4°C with antibody samples and fluorescent antibodies to stain just the cell membrane.

1. The Complete Guide to Antibody Detection
by
Immunofluorescence Technique
—Created by Creative Biolabs

2. Introduction
Immunofluorescence technique can be used in the detection
of hybridoma antibodies of various antigens, such as cellular
antigens (including bacterial and animal cells), viral antigens in
infected cells, and membrane antigens.
Immunofluorescence technique is easy to operate and of high
sensitivity, enabling its ability to directly observe the
advantages of antigen positioning. It possesses important
application value in the screening and identification of
monoclonal antibody (McAb).

3. Equipment and reagents
• Antigen preparation for detection of antibodies: fixed cell
tablet or plate, viable cell suspensions.
• PBS (PH7.2，0.01mol/L)
• McAb samples to be tested
• Cold acetone
• Fluorescein isothiocyanate (FITC) or tetramethyl rhodamine
isothiocynate (TRITC) labeled anti-mouse Ig antibody, etc.
• Fluorescence microscope, magnetic stirrer, centrifuge, water
bath, etc.

4. Indirect immunofluorescence
• Preparation of fixed cell tablet: coverslip with cells (inoculated or unvaccinated
viruses) grew on could be used directly. After washing in PBS, fix it with acetone
of 4 ℃ for 10 minutes. Then dry with air-drying, and seal to preserve at -20 ℃.
Single cell suspension can be spread on the coverclip to make smear. Fix it in the
same way as described above.Feature
• Prepare the cell smear: Apply 10 μl of bacterial suspension (1 × 108 cells/ml) to 7
× 21mm coverslip. After natural drying, fix it with acetone of 4 ℃ for 10-15
minutes and keep it at -20 ℃.
• Moist the coverslip in deionized water, then put it on the back shelf and drop 10-
20ul hybrid supernatant or other samples to be tested on it. Set up positive and
negative control. Incubate in 37 ℃ water bath of 37 ℃ for 0.5-1 hours.

5. Indirect immunofluorescence
• Take out the coverslip and wash it in PBS with a magnetic stirrer for 15
minutes.
• Place the coverslip on a shelf, drop 10-20ul of anti-mouse Ig fluorescent
antibody of working concentration on it. Incubate for 0.5-1 hours at 37 ℃.
• Wash in the same way for 15 minutes. Take out the coverslip, cover the clean
slide with sealing agent that delays fluorescence quenching.
• Observe under a light microscope. If the result is positive, specific
fluorescence could be showed (FITC is in yellow-green fluorescence, TRITC is
in orange-red fluorescence).

6. Indirect immunofluorescence
• The preparation of cell fixed tablet can also be carried out in the wells of the
culture plate. The rest of the steps are the same as above. Turn over the
culture plate and place under fluorescence microscope to observe result
with the same standards.

7. Cell membrane fluorescence staining
• Antibody cannot go through the integrate membrane of a living cell. If the
cells are operated at 4 ℃, the fluorescence staining is limited to the cell
membrane. It must be noted that dead cells often adsorb a large number of
fluorescent antibodies in a nonspecific manner, so the cells should be highly
viable during the test. See below the steps of membrane fluorescence
staining of living cells:
• a. Prepare cell suspension of high activity. Adjust the concentration to 107/ml.
•
• b. Add 100 μl of cell suspension to a small tube (5 × 50 mm). Then add 100μl
of McAb sample to be tested and give it a good mix. Store at 4 ℃ for 30-90
min.

8. Cell membrane fluorescence staining
• c. Wash it with washing solution (900ml of PBS, 50ml of calf serum, 50ml of 4%
NaN3) for twice. Add 1-5ml of washing solution each time, and centrifuge at
1000r/min for 5 minutes. Discard the supernatant, and add 100μl of fluorescent
antibodies. Store at 4 ℃ for 30-90 minutes.
•
• d. Wash it in the same way. Suspend the cells on 20-30ul of solution, which
contains 10% of glycerol and 10-100ug of phenylenediamine per mol. Drop the
suspend ed liquid onto the slide and cap.
•
• e. Check result under a fluorescence microscope immediately.
• f. The cells can also be stained with 1% of paraformaldehyde saline, and then
observe the results immediately or store at least 4 ℃ for one week before
observing.

9. Thank you for watching!
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