Another disadvantage is unavoidable: photobleaching of the fluorochromes. Fluorescent proteins like GFP are also affected by this, but when stored under proper conditions, GFP is detectable even after months in permanent preparations. In contrast, IF fluorochromes lose their intensity faster, which is reflected by rapid bleaching of the sample during microscopy.
Even mounting media with anti-fading agents can only temporarily help here. This is a standard protocol for indirect IF of cultured cells on coverslips with fixation by chemical crosslinkers. A humidified chamber is perfect for the IF procedure and can easily be self-made see Slideshow "How to prepare a humidified chamber".
It prevents drying of the preparation and allows incubation in the dark, which is important in handling fluorochromes and necessary for already existing fluorescent proteins. The volumes are chosen in a way that the coverslips are completely moistened.
Make sure that the sample never fully runs dry. All incubation steps take place at room temperature. Wash the cells twice and use tweezers to carefully place the coverslip with upturned cells into the humidified chamber. Permeabilize with 0. Incubate with the secondary antibody for 1 h, diluted in blocking solution or wash buffer. Take the coverslip gently with tweezers and dip it into dH 2 O to remove residual salts of the wash buffer. Provide a drop of mounting medium on a microscope slide and lay the coverslip with the cells upside down on this drop.
Press the specimen with the tweezers slightly so that the mounting medium is well distributed, without squeezing the sample. The preparation is ready for microscopy after curing. Adjust pH to 7. Talk to our experts. We are happy to answer all your questions and concerns. Do you prefer personal consulting? You will find a more detailed list of local contacts here. April 13, The centerpiece of an IF experiment is a combination of two different components: First, specific antibodies, which are used to form an immune complex to mark the desired molecules — in most cases proteins — in the cell.
Comparing the two IF variants, each of them has different advantages and drawbacks: By coupling the primary antibody with a fluorochrome, direct IF is faster than the indirect version as time-consuming washing and incubation steps are omitted.
Protein A. Protein B. Protein C. Fixative Effect Advantages Disadvantages Chemical crosslinkers Formaldehyde Crosslink proteins via their free amino groups Preserves well cellular morphology.
Good for already present fluorescent proteins. Antigens might also be crosslinked Glutaraldehyde Preserves well cellular morphology. Antigens might also be crosslinked High autofluorescence Organic solvents Methanol Fixation by dehydrogenation and protein precipitation. Cells will simultaneously become permeabilized. Good preservation of cellular architecture. Faster procedure in comparison to chemical crosslinkers.
Strong negative effect on many epitopes. Not suitable for fluorescent proteins. Soluble and lipid components are getting lost. Acetone Less damaging to epitopes. Faster procedure Not suitable for fluorescent proteins. Most cells exhibit Interphase DNA-staining but some cells show condensated chromosomes which get separated in Mitosis asterisks. Threshold for microscopy in direct IF.
Secondary antibody only indirect IF Fixation Permeabilization Blocking Secondary antibody If desired: nucleus staining Unspecific binding of secondary antibody. Threshold for microscopy in indirect IF. Multicolor IF Fixation Permeabilization Blocking Only 1 first antibody Indirect IF: secondary antibody If desired: nucleus staining Compare single stainings to multicolor image: Check for crosstalk of the selected fluorochromes. Check if first antibodies affect each other in epitope binding.
Blocking peptide Fixation Permeabilization Blocking Blocking peptide First antibody Indirect IF: secondary antibody If desired: nucleus staining Check for binding specifity of first antibody to its epitope.
Duration of an IF procedure: approx. For assembly of a humidified chamber you need the following things: a plastic box with lid, an inlay of foam plastic, a slice of parafilm, a robust paper towel, water and tweezers for handling the coverlips. Carefully move the coverslips with tweezers from the culture vessel on the parafilm with upturned cells. Label the the positions of the coverslips and make sure the cells never run dry. Moisten the paper towel and span it tightly on the box. A minute incubation in 0.
View hydrogen peroxide blocking reagent and DAB substrate kit. It can be found in kidney, intestine, osteoblasts, lymphoid tissue and placenta. AP activity is higher in frozen tissue. Levamisole is used for blocking, and is added with the chromogenic substrate. When using a fluorescent label for detection, there is a possibility that the tissue may be autofluorescent, leading to high background.
Tissue fixation may induce autofluorescence, particularly when using aldehyde fixatives eg formalin , which react with amines to generate fluorescent products. Autofluorescence may also be caused by the presence of fluorescent compounds, such as flavins and porphyrins.
These compounds may be extracted from the tissue by the solvents used to generate fixed, dehydrated sections. However, they persist in frozen sections that have been processed using aqueous reagents.
Alternatively, try using frozen tissue sections, or treating tissue with quenching dyes such as pontamine sky blue, Sudan black, trypan blue or FITC block,.
Download the full IHC guide. IHC guide: introduction. Tips for experimental design. IHC guide: sample prep. In general, serum same species as the secondary antibody or bovine serum albumin BSA is used for blocking. Sera and BSA can help to prevent unspecific binding to the many hydrophobic side chains of proteins present in the tissue. If you are staining with multiple antibodies, you need to use blocking serum against all used secondaries.
If BSA is used, the addition of 0. Choose the best blocking solution while working with your negative and positive control samples to set up the threshold of background staining. You can search by either catalog number or antibody name. We understand much of your research is extremely important to the health of the community. As an original manufacturer for its entire catalog of antibodies and proteins, we are here to support you.
Proteintech has five sites globally with full stock inventory available for next day delivery. It is important to ensure the cells do not dry out during this process. Carefully pipette the permeabilization buffer onto the cells and incubate for five minutes. The permeabilization step allows antibodies to access intracellular epitopes and will need optimizing depending on the cell type, antigen, and intended antibody.
There are a number of different permeabilization agents that can be used. For example, Triton X and for very gentle permeabilization, Tween You do not need to permeabilize if the cells were previously fixed in methanol. Once the cells are permeabilized, tap excess buffer off the slides and wash in Phosphate Buffered Saline plus Tween, PBST, on a shaker for five minutes. Repeat with fresh PBST for a total of three wash steps.
Once the washes are complete leave the slides in your wash buffer to avoid them drying out. While your slides are washing, dampen some paper towel and use it to line a slide tray. This will help to keep your slides moist during the next step. Remove excess buffer from your slides and re-draw your PAP pen circles as they may have partially washed off. To prevent non-specific antibody binding, the cells need to be blocked. The type of blocking buffer and incubation time should be optimized for your experiment.
Note; if the cells have been fixed in paraformaldehyde it is common practice to wash with glycine before blocking to quench any remaining fixative. Ideally, the species of serum in the blocking buffer should match the species the secondary antibody was raised in to avoid any cross-reactivity.
Whilst the cells are in blocking buffer, prepare the primary antibodies. After blocking, the cells need to be washed three times for five minutes in PBST as before.
Remove any excess wash buffer and re-draw the PAP pen circles as they may have partially washed off. Place your slides in the line slide tray and add your primary antibody solution. Do this one slide at a time to avoid drying. Place a lid on your tray and carefully transfer it to a fridge to incubate overnight. Wash the slides three times for five minutes in PBST to remove any unbound primary antibody.
If the primary antibodies weren't directly conjugated, incubate the cells for one hour at room temperature with secondary antibodies. Nuclear stains such as Hoechst can be included at this point too. Then wash the slides three times again in PBST.
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