Create mode – the default mode when you create a requisition and PunchOut to Bio-Rad. You can create and edit multiple shopping carts
Edit mode – allows you to edit or modify an existing requisition (prior to submitting). You will be able to modify only the cart that you have PunchedOut to, and won't have access to any other carts
Inspect mode – when you PunchOut to Bio-Rad from a previously created requisition but without initiating an Edit session, you will be in this mode. You cannot modify any Cart contents
Bio-Rad-Antibodies.com relies on third-party cookies to show you pricing, allow you to order online, and connect you to My Bio-Rad. Please amend your browser settings to enable third-party cookies and access this website’s full functionality.
Click here to find out how
Staining intracellular antigens like cytokines can be difficult because antibody-based probes cannot pass easily through the plasma membrane into the interior of the cell. In order to accomplish this, cells should first be fixed in suspension and then permeabilized before adding the antibody. The choice of fixative is an important first step. Formaldehyde and gluteraldehyde create bonds between lysine residues resulting in cross-linked proteins, however gluteraldehyde increases autofluorescence. It is usually used at concentrations of 0.5-4% in PBS depending on the sample. If you are going to store your samples for longer periods of time they should be removed from the fixative after 1-2 hours.
Formaldehyde will not permeabilize the samples so a separate permeabilization step is needed. This allows probes to access intracellular structures while leaving the morphological scatter characteristics of the cells intact. Triton, digitonin and saponin are examples of permeabilization reagents which act by disrupting the cellular membrane. The level of permeabilization is important as epitopes access may require different levels of permeabilization (e.g. cytoplasmic vs nuclear epitopes) and unbound antibody has to be sufficiently washed out of the cells. There are also many commercial kits available today that provide the reagents to carry out both fixation and permeabilization, for example, Leucoperm™ (Figure 28).
Fig. 28. Intracellular staining. Human whole blood (A) was surface stained for CD4 followed by intracellular staining for IFN- gamma, on resting lymphocytes (B) or lymphocytes stimulated with PMA/Ionomycin and treated with monensin (C) prior to staining.
Alcohols are also used as fixatives, typically as a cold 70% solution, that fix by denaturing proteins. The benefits here are that they also permeabilize the cell membrane and are suitable for long term storage at 4oC or -20oC. Epitopes can however be masked by the denaturing process with alcohol fixation, so optimization may be required. Alcohols as a fixative are most commonly used for DNA analysis.
Triton is a trademark of Dow Chemical Company.