FFPE chromogenic IHC protocol Use this workflow for formalin-fixed paraffin-embedded tissue stained with HRP/DAB or another chromogenic detection system. Use this format when: Evaluating protein expression or localization in FFPE tissue sections by brightfield microscopy.Optimize first: Antigen retrieval buffer, retrieval time, primary antibody dilution, blocking, detection chemistry, and hematoxylin counterstain. 1 Bake and deparaffinize slidesBake 4-5 um FFPE sections at 60 degrees C for 30-60 minutes. Deparaffinize in xylene, 2 changes for 5 minutes each. Rehydrate through 100 percent, 95 percent, and 70 percent ethanol, 2-3 minutes each, then rinse in water. 2 Perform antigen retrievalPlace slides in citrate buffer pH 6.0 or Tris-EDTA buffer pH 9.0. Heat near boiling for 10-20 minutes using a pressure cooker, microwave, steamer, or water bath. Cool slides in buffer for 20 minutes, then rinse in TBS or PBS. 3 Block endogenous activityFor HRP detection, incubate slides in 3 percent hydrogen peroxide for 10 minutes to quench endogenous peroxidase. Rinse 3 times in buffer. 4 Block nonspecific bindingApply protein block, 5 percent normal serum, or 1-5 percent BSA for 20-60 minutes at room temperature. Drain but do not rinse if the detection system recommends leaving blocking protein on the section. 5 Add primary antibodyDilute BRCA1 Polyclonal Antibody in antibody diluent. Start with the datasheet-recommended IHC dilution or test 1:50, 1:100, 1:250, and 1:500. Apply 100-200 uL per section and incubate 1 hour at room temperature or overnight at 4 degrees C in a humidified chamber. 6 Wash after primary antibodyWash 3 times for 5 minutes each in TBS-T or PBS-T. Keep slides covered with buffer and do not let tissue dry. 7 Apply secondary or polymer detectionApply HRP polymer, biotinylated secondary plus streptavidin-HRP, or another detection reagent according to the system instructions. Typical incubation is 20-30 minutes at room temperature. Wash 3 times. 8 Develop chromogenApply DAB or other chromogen for 1-10 minutes while monitoring signal under a microscope. Stop development by rinsing in water. 9 Counterstain and mountCounterstain with hematoxylin for 15 seconds to 2 minutes, blue in running tap water or bluing reagent, dehydrate through ethanol, clear in xylene, and mount with permanent mounting medium. Retrieval optimizationIf signal is weak, compare pH 6 and pH 9 retrieval before dramatically increasing antibody concentration.Negative controlUse no-primary, isotype control when appropriate, and known negative tissue to evaluate nonspecific staining.
Frozen tissue IHC protocol Use this workflow for frozen sections when antigen preservation is more important than FFPE morphology. Use this format when: The epitope is fixation-sensitive, lipid-rich tissue is used, or frozen-section staining is preferred.Optimize first: Fixation method, section thickness, drying time, blocking, primary antibody dilution, and tissue autofluorescence or endogenous enzyme activity. 1 Prepare frozen sectionsCut 5-10 um cryosections and mount onto charged slides. Air dry 10-30 minutes at room temperature. Store slides cold if staining later. 2 Fix sectionsFix with cold acetone for 10 minutes at -20 degrees C, cold methanol for 5-10 minutes, or 4 percent paraformaldehyde for 10-15 minutes depending on the target. Rinse gently in PBS or TBS. 3 Block endogenous activity if neededFor HRP-based detection, quench endogenous peroxidase with 0.3-3 percent hydrogen peroxide for 10 minutes. For biotin-based detection, consider avidin/biotin blocking when tissue has high endogenous biotin. 4 Block nonspecific bindingBlock 20-60 minutes with 5 percent normal serum or 1-5 percent BSA in PBS/TBS. Use serum from the secondary antibody host species when practical. 5 Add primary antibodyDilute BRCA1 Polyclonal Antibody in antibody diluent. Start with the datasheet-recommended frozen IHC dilution or test 1:50 to 1:500. Apply 100-200 uL per section and incubate 1 hour at room temperature or overnight at 4 degrees C. 6 Wash and detectWash 3 times for 5 minutes. Apply secondary antibody, polymer detection, or fluorescent secondary antibody according to the detection system. Incubate 30-60 minutes. 7 Develop or mountFor chromogenic detection, apply substrate until signal develops, rinse, counterstain, and mount. For fluorescent detection, protect from light, counterstain with DAPI if needed, and mount with antifade medium. MorphologyFrozen sections usually preserve antigenicity better but morphology can be less crisp than FFPE tissue.Section adhesionUse charged slides and avoid harsh washing if sections lift from the slide.
Fluorescent IHC protocol Use this workflow for fluorescent staining of tissue sections when colocalization or multiplex imaging is needed. Use this format when: Tissue localization is needed with fluorescent readout, multiple markers, or confocal microscopy.Optimize first: Autofluorescence control, antigen retrieval, fluorophore choice, antibody host species, and mounting medium. 1 Prepare and retrieve tissueFor FFPE tissue, deparaffinize, rehydrate, and perform antigen retrieval. For frozen tissue, fix according to target requirements. Rinse in PBS or TBS. 2 Reduce autofluorescenceTreat autofluorescent tissues with a compatible quenching reagent when needed. Avoid quenchers that interfere with the planned fluorophores. 3 Permeabilize and blockUse 0.1-0.3 percent Triton X-100 for intracellular access when compatible. Block with 5 percent normal serum or 1-5 percent BSA for 30-60 minutes. 4 Add primary antibodyDilute BRCA1 Polyclonal Antibody in blocking buffer. Start with the datasheet-recommended IHC/IF dilution or test 1:100, 1:250, and 1:500. Apply 100-200 uL per section and incubate 1-2 hours at room temperature or overnight at 4 degrees C. 5 Wash and add fluorescent secondaryWash 3 times for 5 minutes. Add cross-adsorbed fluorescent secondary antibody at 1:500-1:1000 for 45-60 minutes protected from light. 6 Counterstain and mountWash 3 times. Add DAPI if desired. Mount with antifade medium and coverslip without bubbles. 7 Image and controlAcquire no-primary, single-color, and full-stain controls. Use identical image settings for samples that will be compared. Multiplex tissue stainingFor multiple primary antibodies, confirm species compatibility or use directly conjugated primaries or sequential staining.AutofluorescenceTissue autofluorescence can resemble true signal. Always compare to no-primary controls.
IHC optimization protocol Use this workflow when an antibody or tissue type needs condition screening before final staining. Use this format when: Signal is weak, background is high, staining is inconsistent, or the antibody is being validated in a new tissue.Optimize first: Positive tissue, retrieval pH, antibody dilution, incubation time, detection strength, and blocking strategy. 1 Choose control tissueUse a known positive tissue or cell pellet and a known negative tissue when available. Include the experimental tissue only after positive control staining is working. 2 Build a retrieval matrixTest no retrieval, citrate pH 6.0, and Tris-EDTA pH 9.0 when tissue availability allows. Use the same section thickness and slide type for all conditions. 3 Test antibody dilutionDilute BRCA1 Polyclonal Antibody across a small matrix such as 1:50, 1:100, 1:250, and 1:500, or follow datasheet starting ranges. Keep retrieval and detection constant while testing dilution. 4 Compare incubation conditionsIf signal is weak, compare 1 hour at room temperature with overnight at 4 degrees C. Longer incubation can improve weak signal but may increase background. 5 Adjust detection strengthIf signal remains weak, increase polymer incubation within the detection system limits, use amplification, or extend chromogen development while monitoring background. 6 Reduce backgroundIncrease wash time, reduce antibody concentration, change blocking buffer, shorten chromogen development, or add detergent to wash buffer if compatible with the tissue and detection system. 7 Lock the protocolOnce the best condition is chosen, stain all comparative samples together using the same retrieval, antibody dilution, incubation time, detection system, and development time. One change at a timeChange one major variable at a time when troubleshooting so the cause of improvement is clear.DocumentationRecord lot numbers, retrieval buffer, heating method, antibody dilution, incubation time, detection system, and imaging settings.
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