The standard procedure for intracellular staining with FIX & PERM® gives optimal results for most antigens. A modification using precooled absolute methanol has been shown to give better results for certain cell cycle antigens such as Ki-67 and PCNA when using FITC conjugated antibodies. The methanol step is not recommended when using R-PE conjugated antibodies.
These staining protocols are intended for use directly with the cell suspension to be analyzed. Other lysing solutions should not be used prior to the use of FIX & PERM® reagents.
FIX & PERM® reagents are designed for use with all commercially available flow cytometers. Alignment and compensation should be performed according to the manufacturer's instructions. Typical scatter and staining patterns for a number of applications are shown on the following pages.
Flow cytometric analysis with monoclonal antibodies have been restricted primarily to cell surface molecules. Intracellular structures such as cytoplasmic or nuclear enzymes, oncoproteins, cytokines, immunoglobulins etc. were largely excluded from such studies.
Also excluded from such flow cytometric studies were cytoplasmic localizations of well established membrane molecules such as CD3 and CD22.
FIX & PERM® reagents allow intracellular antigen analysis with the same ease as surface antigens. The only prerequisite is the availability of suitable monoclonal antibody conjugates.
Most of the commercially available monoclonal antibody conjugates can be used with the FIX & PERM® reagents. Some determinants are sensitive, however, to the fixation step involved. This, and the optimal fixation time, may have to be determined experimentally for each antibody conjugate.
COMBI-IC® and CYTO-IC® reagents offered by CALTAG/AN-DER-GRUB have been determined to perform optimally when used with FIX & PERM®.
A frequently asked question is whether indirect staining can be performed with FIX & PERM®. The answer is yes with the addition of another washing step. It is not necessary to add additional Reagent B when adding the second step reagent. Higher background staining can be encountered with indirect assays and it is usually preferable to use directly conjugated antibodies where available.
FIX & PERM® reagents should be stored at room
temperature. They are stable for the period shown on the package label when
stored as directed. Do not use reagents if a precipitate forms or discoloration
occurs. All antibody combinations should be stored in the dark at 2-8°C.
R-PE
conjugates should never be frozen.
Reagent A of the FIX & PERM® kit contains formaldehyde which is toxic, allergenic and a suspected carcinogen. Avoid contact with eyes, skin and clothing. All antibodies contain sodium azide as preservative.
These reagents are intended for research or investigational use only. They are not intended for therapeutic or in vitro diagnostic use.