Please take note of the general remarks below before using the immuno incubation protocol for Light Microscopy.
Recommended Incubation Solution:
PBS (10 mM phosphate buffered saline), pH 7.4
0.1-0.2% AURION BSA-c™
check the pH and adjust to 7.4 if necessary
This buffer system is recommended both for secondary antibody reagents as well as for protein A, protein G and streptavidin reagents. Buffers are preferably prepared fresh and can be used for periods up to two weeks.
AURION Ultra Small Immunogold Reagents are the reagents of choice for the localization of extracellular and intracellular antigens in light microscopy.
The degree of penetration of immuno reagents into the cell interior depends on size of the reagents, specimen characteristics and (aldehyde)fixation. For the localization of intracellular antigens AURION Ultra Small Immuno gold Reagents occasionally require a permeabilization with e.g. Triton-X-100® in the first washing step immediately following fixation. In this way difficult to access intracellular antigens can be localized.
Living cells are preferably incubated at 0-4°C or in the presence of 0.05-0.2% NaN3 in order to prevent internalization of reagents.
Monolayers on coverslips are easily incubated using 6-well culture plates (Falcon, Nunc etc.) The glass coverslips are placed in the well and covered with incubation media (approximately 100 µl). During washing the coverslips are covered with 2 ml of washing medium and left on a rocking table.
Cell suspensions are gently pelleted after each incubation step. The pellets are resuspended in the medium used in the next step and the centrifuge tube containing the suspension is left on a rocking table.
On occasion efficient background suppression is obtained by using 1-10% heat inactivated Human AB-serum as additive to the incubation buffer.
Should a permeabilization step be necessary the following procedure may be employed:
Triton-X-100® is added in a final concentration of 0.1 – 0.5% to the washing buffer used immediately following fixation. This permeabilization step should last between 10 and 20 minutes while shaking gently.
NOTE: The use of more or less apolar fixatives (e.g. based on methanol, acetone, ethanol) already infers a limited degree of permeabilization to specimens as part of the lipid is removed.
Procedures for marking
Procedures can e.g. be found in the CRC Press-edition “Immunogold Labeling in Cell Biology”, A.J. Verkley & J.L.M. Leunissen eds., (1989), Boca Raton, Florida. In addition the issues of the Academic Press edition “Colloidal Gold”, M.A. Hayat ed., (1989), San Diego, California are highly recommended.