FAQs Immunochemistry General Questions VECTASTAIN ABC Kits Immunofluorescence VECTASHIELD Antifade Mounting Media General Questions Could you please describe what would be an appropriate negative control for IHC that can be used to help validate tissue staining results? A commonly used negative control is omission of the primary antibody. While this control addresses whether the secondary antibody reagents are a source of staining, inadvertent binding of the primary antibody to the tissue can occur. Various tissue elements such as Fc receptors and charged molecules may bind the primary antibody non-specifically. Simply omitting the primary antibody as a negative control would miss potential false positive staining by this means. A few “publication worthy” negative controls for IHC are listed below: 1) Preabsorption of primary antibody with the immunogen used to generate the antibody can be employed. The working dilution of the primary antibody and an optimized concentration of the immunogen are incubated together for a period prior to application to the specimen. Lack of staining would indicate specificity of the primary antibody to the target antigen in solution. Positive staining using this method, however, may indicate lack of specificity of the primary antibody and/or the primary antibody is being bound by tissue elements. To rule out the latter, this control can be used in combination with suggestion no. 2 below. Note that adsorption controls are not always feasible or practical depending on the cost or source of the immunogen. 2) Use of an isotype control (e.g. “non-immune” mouse IgG), matched to that of the primary antibody and applied at the same protein concentration as the primary antibody, is probably the most widely used negative control. This control addresses whether tissue elements are inadvertently binding immunoglobulin from the same species as the primary antibody, in addition to non-specific binding from the secondary detection reagents. In most cases, use of a sub-class of isotype immunoglobulin (e.g. mouse IgG2a or IgG2b) is not required. Note that the use of pre-immune immunoglobulin, obtained prior to immunization, could also be used. However, it is very unusual for commercial vendors to offer pre-immune immunoglobulin. 3) Substitution of the primary antibody with an “irrelevant antibody” is also a suitable negative control. The term “irrelevant” refers to a primary antibody of the same isotype as the specific primary antibody (i.e. mouse IgG) and applied at the same concentration, that is known not to bind to a target in the tissue specimen. An example would be an anti-cytokeratin antibody on smooth muscle tissue. As with negative control no. 1 above, lack of staining indicates tissue elements are not binding this isotype of immunoglobulin. 4) In some cases, the target antigen can be removed from the tissue specimen as a sort of “knock-out” preparation. Once the target antigen has been removed, the complete assay is run to determine lack of staining. One method to remove the target antigen is using defined enzyme digestion. Examples include the use of a collagenase if the target antigen is collagen, or hyaluronidase if the target antigen is hyaluronic acid. Of course, there are limitations to this approach, however, variations of this “deletion” or “knock-out” approach would be valid negative controls. For my IHC application, I intend to apply a mouse IgG primary antibody on rat tissue sections. Should I use the Vector Mouse on Mouse detection kit? Vector Laboratories’ Mouse on Mouse detection kits (M.O.M.®) are designed for the detection of mouse IgG primary antibodies on mouse tissue sections. For detecting mouse primary antibodies on rat tissue, we offer a selection of anti-mouse, rat adsorbed secondary detection reagents. These are intended to be used for this application and would generate the most optimal signal to noise staining ratio. We offer a biotinylated anti-mouse IgG, rat adsorbed secondary antibody (Cat. No. BA-2001) for use with VECTASTAIN® ABC kits or avidin and streptavidin enzyme conjugates. Alternatively, we offer an ImmPRESS® HRP polymer anti-mouse IgG, rat adsorbed detection kit (Cat. No. MP-7422) for a convenient, one-step IHC methodology. How long can I store VECTABOND® working solution once diluted in acetone? VECTABOND Reagent is supplied as a 7 ml concentrate that is diluted in acetone immediately prior to use. Once diluted in acetone, as per the instructions for use, it is recommended that the working solution be used as soon as possible. Storage of this working solution is not practical beyond a few hours as the chemicals will start to break down and the solution will turn a yellowish color. Once this has happened, slide coating will not be optimal and the working solution should be discarded. I am performing IHC on tissue sections using a mouse IgG primary antibody. My detection system is a biotinylated anti-mouse IgG secondary antibody, a VECTASTAIN® Elite ABC peroxidase kit (PK-6100), in Make a fresh working solution of DAB substrate per instructions. Place a small volume (~1 mL) of this DAB substrate into a clean glass test tube. To this 1 ml aliquot, add one drop (~50 µL) of Reagent B only from the VECTASTAIN® Elite ABC kit. If the HRP enzyme is active and the DAB reacts with it, an immediate color change will be observed. This indicates the end detection reagents are working appropriately. I am using Methyl Green (H-3402) as a counterstain for my IHC, but the stain is a little weak. What suggestions do you have to increase the staining intensity? Methyl Green counterstain does take a little bit of optimization in some applications. We would suggest heating a volume of the counterstain (~300 ml) to 60 °C and add to a Coplin jar or similar glass staining rack, and submerse the slides. This approach facilitates a better uptake of stain into the section compared with placing the slide on a heated surface and placing drops of stain onto the section. Following the incubation time (~3-5 min), remove slides and wash in tap water. Omit the acetone, acetic acid rinse step described in the instructions and move the slides directly into 95% ethanol for the dehydration, clearing and mounting process. This methodology will retain more stain in the nuclei and hence produce greater intensity. I have a bottle of the antigen unmasking solution, H-3300. It has been stored in the fridge for a few weeks. When I opened it, it contained some precipitate. Do you know what this precipitate is, and Product H-3300 is supplied as a highly concentrated (100x) salt solution. It is recommended to be stored in the fridge. In some cases, over time with cold storage, some salts may come out of solution and appear as particulate or precipitated material. Our recommendation is to gently warm the bottle in a warm water bath to re-dissolve the precipitate. Usually 30 min at 35 °C would be sufficient. Once re-dissolved, an aliquot can be drawn from this solution and diluted according to the instructions. Re-dissolving the precipitate maintains the desired pH and salt concentration for optimal performance. In some IHC detection reagent instructions, the last step states “dehydrate, clear and mount”. What does this mean? In a manual IHC application, these steps involve submersion of the slides into a graded ethanol series, immediately followed by submersion into a miscible solution (xylene or alternative), followed by placement of a coverslip over the preparation with a suitable mounting medium. These steps preserve the specimen for subsequent visualization, imaging and archiving. From our experience, we suggest moving the slides in a slide rack through a series of dishes, commencing dehydration at 95% ethanol (2 changes for 2 min ea.), then into 100% ethanol (2x 2 min ea.), then into clearing solution (3x 2 min ea.), followed by mounting (coverslipping). My enzyme substrate precipitates at the site of localization, but it disappears after the dehydration through alcohol. How can I prevent the enzyme substrate from fading? We have found that some substrates tend to be a little soluble in lower ethanol grades (i.e. 70% EtOH) during the dehydration step. From our experience, we suggest moving the slides in a slide rack through a series of dishes, commencing dehydration at 95% ethanol (2 changes for 2 min ea.), then into 100% ethanol (2x 2 min ea.), then into clearing solution (3x 2 min ea.), followed by mounting (coverslipping). We recommend diluting 95% ethanol directly from 100% ethanol, instead of using 95% ethanol from a vendor. The ethanol that we use for dehydration is obtained from VWR analytical. The catalog # is BDH1156-4LP (4 L size). It is a mixture of ethanol, methanol (~94-96%) and isopropanol (~4-6%). What are some considerations when applying a counterstain for immunohistochemistry (IHC)? The use of a counterstain for IHC is optional and should only be applied if it may be helpful. The idea of using a counterstain is to provide a contrasting color to tissue structures and architecture, compared with the specific target staining generated by the substrate precipitate, to help in the visualization of the overall morphology and cell type in a heterogeneous specimen. As an example, the combination of a brown DAB substrate specific stain with a blue nuclear hematoxylin counterstain is probably the most widely used in standard IHC. A further consideration would be controlling the intensity of the counterstain, usually through incubation time, to ensure it does not obscure the specific substrate stain, thereby generating a false negative result. With regard to this last point, if you are trying to visualize a weakly expressed, possibly transient upregulated target antigen or indeed an antigen of unknown expression in your preparation, omitting a counterstain would be recommended until a baseline (validated positive expression) of specific staining is established. We provide several nuclear counterstain options, and resources such as a counterstain/substrate compatibility table that should help in the selection of an appropriate combination for your application. What are the components in the antigen unmasking solution, citrate buffer pH 6, H-3300? The stock solution of H-3300 is supplied as 1M citrate. Once diluted according to the instructions, the working concentration would be 0.01 M citrate. It is essentially a saturated concentrated salt solution. What is the capacity of the ImmEdge Hydrophobic Barrier PAP Pen (H-4000)? The volume of the ImmEdge Pen (H-4000) is about 6 mL. We have not characterized how many times it can be used or overall length of the barrier due to variability in its application. VECTASTAIN ABC Kits Can I perform double staining for two different antigens on the same section using two mouse primary antibodies with the same VECTASTAIN ABC Mouse IgG Kit? Yes. We do offer complete protocols, helpful tips as well as enzyme substrate combination suggestions and images on our website for this application at the following link: https://vectorlabs.com/guides/multiple-antigen-labeling-guide. Essentially the staining is performed sequentially (one stain after the other) from primary antibody right through to substrate color development using two different enzyme substrates. How do I choose which VECTASTAIN ABC Kit to use? We offer a choice of either peroxidase or alkaline phosphatase based VECTASTAIN ABC kit detection systems. Selection of which enzyme system to use would be the first step. Most IHC applications involve detecting an unconjugated primary antibody. Consider the species in which the primary antibody is raised (e.g. rabbit), and then match to to corresponding species-specific VECTASTAIN ABC kit (e.g. VECTASTAIN ABC Rabbit IgG kit). If you already have a biotinylated target (i.e. biotinylated secondary antibody or primary antibody), then a standard VECTASTAIN ABC kit containing just the avidin/biotinylated enzyme reagents would be required. Note that only the VECTASTAIN Universal Elite PLUS ABC kit (PK-8200) contains a substrate. What is the difference between the VECTASTAIN ABC regular peroxidase kits (PK-4000 series) and the VECTASTAIN Elite ABC peroxidase kits (PK-6000 series)?Can the reagents from a regular ABC kit be inte The key difference between the VECTASTAIN ABC regular kits and the VECTASTAIN Elite ABC kits is sensitivity. The VECTASTAIN Elite ABC kits are 5x greater in sensitivity than the regular kits. This increased sensitivity allows for further dilution of a potentially expensive primary antibody and detection of weaker (lower abundance) expressed target antigen. This difference in sensitivity is achieved through slightly different chemistry in the VECTASTAIN Elite ABC reagents that introduces more peroxidase enzyme and hence generates a greater reaction with the substrate and color deposition at the site of antigen localization. The sera and secondary antibodies in the VECTASTAIN regular and Elite ABC kits are the same and can be used interchangeably. However Reagent A (avidin) and Reagent B (biotinylated enzyme) are specific for the kit and are not interchangeable. What is the stability of the working solutions of the VECTASTAIN ABC kits once made up according to the instructions? We recommend using the working solution of the diluted ABC reagent (mixing Reagent A and Reagent B together) within 24 hour of being made to retain maximum enzyme activity and performance. This will allow for comparison of staining results between assays. In the species-specific kits, diluted (working solution) of the sera and biotinylated secondary antibodies can be kept in the fridge for up to 1 week. Note that Ready-To-Use (RTU) formats of the VECTASTAIN ABC reagents are offered for greater convenience and stability of the working solutions. Immunofluorescence - VECTASHIELD Antifade Mounting Media How quickly can I view sections after mounting with VECTASHIELD® Vibrance (catalog # H-1700)? Is it recommended to wait one hour? Sections can be viewed immediately after mounting with VECTASHIELD® Vibrance, but one hour is recommended for optimal signal intensity. Can VECTASHIELD® be used for super resolution microscopy? Of the many variations of super resolution microscopy (SRM) currently being used, investigators have found the properties of product H-1000 (VECTASHIELD), to be advantageous in stochastic optical reconstruction microscopy (STORM) and structured illumination microscopy (SIM). Refer to the following published references for further information: 1) Olivier, N., et al (2013) Simple buffers for 3D STORM microscopy. Biochemical Optics Express 4:885-899. 2) Wegel., et al (2016) Imaging cellular structuires in super-resolution with SIM, STED and localisation microscopy: A practical comparison. Scientific Reports 6:27290. Do I need to dehydrate the tissue sections before applying VECTASHIELD? No form of tissue dehydration (e.g. air drying or ethanol exposure) is required nor recommended when applying VECTASHIELD. From our experience, the most optimal antifade actions of VECTASHIELD are obtained when the preparation is removed from the final buffer/water rinse, kept slightly wet/moist and then coverslipped with a small volume (25-50 uL) of VECTASHIELD. Do I need to seal the perimeter of the coverslip after I apply VECTASHIELD? That depends upon how long you wish to retain the slides and which VECTASHIELD formulation you are using. If you are using one of our non-setting VECTASHIELD products such as H-1000 or H-1200, then we suggest sealing the coverslip with plastic sealant or nail polish if you intend to keep the slides beyond a day or so. If you are using one of our setting/curing formulations such as VECTASHIELD HardSet or Vibrance, then in most cases when using thin cut (<10 um) tissue sections or cell monolayers, no sealing of the coverslip is requuired. Does the coverslip need to be sealed with nail polish or similar compound after mounting? Similar to other non-setting media, we recommend you to seal the coverslip perimeter if the intention is to store the slides for a period of time. Does VECTASHIELD contain glycerol? All of the VECTASHIELD formats we currently offer which includes regular non-setting VECTASHIELD, HardSet and Vibrance formulations each contain a percentage of glycerol.