Fluorescent Labeled Lectin Procedure

Fluorescent Labeled Lectin Procedure

(FITC, TRITC, Texas Red®)

Absorption and Emission

Absorption/Excitation Rate Emission Max.
FITC 492 nm 517 nm
TRITC 554 nm 570 nm
Texas Red® 596 nm 615 nm

Tissue Sections

1. Wash and block the tissue section.  Do not use serum products, they contain glycoproteins which may lead to high levels of non specific background.  After blocking, rinse briefly with the appropriate Buffer.

2. Using the appropriate Buffer, dilute the Fluorescent Labeled Lectin to the desired concentration of 20-100 mg/ml.

3. Incubate the tissue section with the Fluorescent Labeled Lectin for 30 minutes in a moist chamber.

4. Wash the tissue section with the Buffer three times.

5. Examine the tissue section using a Fluorescent microscope.  Use the appropriate filter.
(Ref. M. Immbar et. al., (1973). Intl. Journal of Cancer, 12, 93-99.)

Cell Suspension           

1. Wash the cells with the appropriate Buffer.

2. Collect the cells by centrifugation.

3. Using the appropriate Buffer, dilute the Fluorescent Labeled Lectin to a concentration of 100 mg/ml.

4. Incubate approximately 1×106 cells with 1 ml of the diluted Fluorescent labeled Lectin for 15 minutes at room temperature or in a 37°C water bath.

5. Wash cells with the Buffer three times by means of centrifugation.

6. Examine cells, with or without fixation using a Fluorescent microscope.  Use the appropriate filter.

(Ref. K. Phiss. (1977).Experimental Pathology, 14, S15.)

                  Fluorochromes must be protected from light.  Perform incubation, when practical, in a dark room or covered in foil

NOTES: Inhibition of lectin binding may be accomplished by using one of the following procedures:

A. Preincubate the section or cells with the inhibitory carbohydrate for 30-60 minutes at room
temperature before incubating with Fluorescent Labeled Lectin.  Complete inhibition may
not  occur.

B. Preincubate the diluted Fluorescent Labeled Lectin with the inhibitory carbohydrate for
30-60 minutes at room temperature before applying to the section or cells.