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 CASP7 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 CASP7 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 CASP7 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 CASP7 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.
Protein immunoprecipitation protocol Use this workflow to enrich a target protein from cell or tissue lysate using an antibody and protein A/G magnetic or agarose beads. Use this format when: Pulling down a target protein for Western blot, mass spectrometry, enzymatic assay, or interaction analysis.Optimize first: Lysis buffer stringency, lysate amount, antibody amount, bead amount, binding time, wash stringency, and elution method. 1 Prepare cold lysis bufferUse non-denaturing IP lysis buffer such as 20-50 mM Tris or HEPES pH 7.4-7.6, 150 mM NaCl, 1 percent NP-40 or Triton X-100, 1 mM EDTA, and fresh protease inhibitors. Add phosphatase inhibitors for phospho or PTM targets. 2 Lyse sampleUse 0.5-2 mg total protein per IP as a starting range. Lyse cells on ice for 15-30 minutes with periodic mixing. For tissue, homogenize gently in cold lysis buffer. Clarify lysate at 12,000-16,000 x g for 10-15 minutes at 4 degrees C. 3 Pre-clear lysateAdd 20-30 uL washed protein A/G beads per IP to lysate and rotate 30-60 minutes at 4 degrees C. Collect supernatant and discard pre-clear beads to reduce nonspecific binding. 4 Bind antibody to targetAdd 2-5 ug CASP7 Polyclonal Antibody per 0.5-1 mg lysate as a starting point, or follow the datasheet. Rotate 2-4 hours at 4 degrees C, or overnight for low-abundance targets. 5 Capture immune complexesAdd 20-40 uL washed protein A/G magnetic or agarose beads per IP. Rotate 1-2 hours at 4 degrees C. Use protein A, protein G, or A/G according to antibody species and isotype. 6 Wash beadsWash beads 3-5 times with 0.5-1 mL cold lysis buffer. For high background, add one higher-salt wash such as 300-500 mM NaCl if the interaction or epitope can tolerate it. Keep beads cold and minimize bead loss. 7 Elute targetFor Western blot, add 20-40 uL 1x or 2x SDS sample buffer and heat 70-95 degrees C for 5-10 minutes depending on target stability and bead type. For native elution, use low pH glycine or competing peptide if compatible. 8 Analyze eluateRun input, flow-through if needed, wash control, IgG control, beads-only control, and IP eluate. For WB detection, use a secondary antibody strategy that minimizes IgG heavy-chain and light-chain interference. Antibody compatibilityWB validation does not guarantee IP performance. IP requires antibody binding under native or partially native conditions.Heavy chain interferenceUse crosslinked antibody beads, light-chain-specific secondary antibody, native elution, or antibodies from different species when heavy chain overlaps the target size.
Co-IP protocol Use this workflow to preserve and test protein-protein interactions during immunoprecipitation. Use this format when: Pulling down a bait protein and detecting associated partner proteins.Optimize first: Mild lysis conditions, salt concentration, detergent choice, lysate freshness, wash stringency, and reciprocal IP controls. 1 Use mild lysis conditionsPrepare cold mild lysis buffer such as 20-50 mM Tris or HEPES, 100-150 mM NaCl, 0.2-0.5 percent NP-40 or digitonin, 1 mM EDTA, and fresh inhibitors. Avoid harsh detergents and high salt unless the interaction is strong. 2 Lyse gentlyUse fresh cells when possible. Lyse on ice for 15-30 minutes with gentle mixing. Clarify lysate at 10,000-14,000 x g for 10 minutes at 4 degrees C. Avoid sonication unless necessary because it can disrupt complexes. 3 Pre-clearPre-clear lysate with 20-30 uL beads for 30-60 minutes at 4 degrees C. Transfer the supernatant to a new tube. 4 Add bait antibodyAdd 2-5 ug CASP7 Polyclonal Antibody per IP if it is the bait antibody. Rotate 2-4 hours at 4 degrees C. For weak interactions, overnight incubation can help but may increase nonspecific binding. 5 Capture complexesAdd 20-40 uL protein A/G beads and rotate 1-2 hours at 4 degrees C. Keep all steps cold. 6 Wash gentlyWash 3-4 times with mild lysis buffer. If nonspecific bands are high, increase salt stepwise to 200-300 mM NaCl or reduce detergent exposure based on complex stability. 7 Elute and detectElute with SDS sample buffer for Western blot. Detect bait and suspected partner proteins on separate blots or after stripping when appropriate. 8 Confirm interactionInclude IgG control, beads-only control, input lysate, and reciprocal Co-IP when possible. Validate that the partner signal is enriched in the bait IP compared with controls. Complex preservationCo-IP success depends heavily on keeping the complex intact. Use fresh lysate, cold buffers, and mild washing first.Nuclease optionFor DNA- or RNA-mediated associations, add nuclease controls when needed to distinguish direct protein interaction from nucleic-acid bridged association.
Crosslinked IP or chromatin-associated target protocol Use this workflow as a general crosslinked IP starting point for chromatin-associated proteins. For full ChIP workflows, see the dedicated ChIP protocol. Use this format when: A chromatin-associated protein or complex needs crosslink-stabilized enrichment before downstream analysis.Optimize first: Crosslinking time, chromatin fragmentation, antibody amount, wash stringency, reversal conditions, and DNA/protein recovery method. 1 Crosslink sampleTreat cells with 1 percent formaldehyde for 10 minutes at room temperature as a starting condition. Quench with 125 mM glycine for 5 minutes. Wash cells with cold PBS containing inhibitors. 2 Prepare nuclei or lysateCollect cells and prepare nuclei or chromatin-enriched lysate using a compatible buffer. Keep samples cold and include protease inhibitors. 3 Fragment chromatin or complexesSonicate or digest to produce the fragment size needed for the downstream assay. For ChIP-qPCR, 200-800 bp DNA fragments are often used. Confirm shearing before large experiments. 4 Clarify and pre-clearClarify lysate and pre-clear with blocked protein A/G or ChIP-compatible beads for 30-60 minutes at 4 degrees C. 5 Add antibodyAdd 2-10 ug CASP7 Polyclonal Antibody per IP depending on target abundance and chromatin amount. Rotate overnight at 4 degrees C for chromatin-associated targets. 6 Capture and washAdd blocked beads and rotate 1-2 hours. Wash sequentially with low-salt, high-salt, LiCl or detergent wash, and TE buffer when compatible with the downstream method. 7 Elute and reverse crosslinksElute complexes and reverse crosslinks, commonly at 65 degrees C for 2-6 hours or overnight depending on protocol. Treat with RNase and proteinase K if recovering DNA. 8 Purify and analyzePurify DNA or protein as required. Analyze by qPCR, sequencing library preparation, Western blot, or other downstream method. Dedicated ChIP pageFor optimized native and crosslinking ChIP details, see the dedicated ChIP protocol.CrosslinkingOver-crosslinking can reduce antibody access and recovery. Start with mild crosslinking and optimize.
RNA immunoprecipitation protocol Use this workflow to enrich RNA-associated proteins or protein-bound RNA while preserving RNA integrity. Use this format when: Studying RNA-binding proteins, protein-RNA complexes, or RNA associated with a target protein.Optimize first: RNase-free handling, lysis conditions, antibody amount, salt concentration, wash stringency, and RNA purification. 1 Prepare RNase-free workspaceUse RNase-free tubes, filter tips, buffers, and gloves. Add RNase inhibitor to lysis, wash, and binding buffers when compatible. 2 Lyse cells gentlyLyse cells in RIP lysis buffer containing mild detergent, physiological salt, protease inhibitors, and RNase inhibitor. Use fresh lysate when possible. Clarify at 12,000-16,000 x g for 10 minutes at 4 degrees C. 3 Pre-clear lysatePre-clear lysate with washed beads for 30-60 minutes at 4 degrees C. Transfer supernatant to a new RNase-free tube. 4 Add antibodyAdd 2-10 ug CASP7 Polyclonal Antibody per RIP depending on target abundance. Rotate 2-4 hours or overnight at 4 degrees C. Include IgG control and input RNA controls. 5 Capture complexesAdd 20-40 uL protein A/G beads that have been washed and blocked with BSA, yeast tRNA, or other compatible blocking reagent. Rotate 1-2 hours at 4 degrees C. 6 Wash under RNase-free conditionsWash 4-6 times with cold RIP wash buffer. Increase salt only if nonspecific RNA background is high and the interaction can tolerate it. 7 Recover RNADigest protein or reverse crosslinks if used, then purify RNA using phenol/chloroform, column purification, or magnetic bead cleanup. Include DNase treatment when downstream qPCR could detect DNA contamination. 8 Analyze RNAAnalyze enriched RNA by RT-qPCR, sequencing, or other downstream assay. Normalize to input and compare to IgG control. RNase controlRNA loss is often caused by RNase contamination. Use RNase inhibitor and work quickly on ice.Input controlSave 1-10 percent input before IP for normalization.
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