TSA DNP kits Protocol-in-brief: in situ hybridization Preparation: TNT Wash Buffer: 0.1 M Tris-HCl, pH 7.5 0.15 M NaCl 0.05% Tween 20 TNB Blocking Buffer: 0.1M Tris-HCl, pH 7.5 0.15 M NaCl 0.5% Blocking Reagent (w/v), supplied in kit or purchased separately FP1020: 3 g. blocking reagent FP1012: 20 g. blocking reagent Add Blocking Reagent slowly in small increments to buffer while stirring. Heat gradually to 55 °C with continuous stirring to completely dissolve the Blocking Reagent. This may take up to several hours. Aliquot and store at -20 °C for long term use. Discard any unused blocking buffer which has been stored for more than 24 hours at room temperature. DNP Stock Solution The DNP Amplification Reagent is supplied as a solid. Reconstitute as indicated in product manual. Use molecular biology grade or HPLC-grade DMSO. The DNP Amplification Reagent Stock Solution, when stored at 4° C, is stable for at least 6 months. (DMSO freezes at 4° C. Thaw the Stock Solution before each use.) Catalog number NEL746A NEL746B NEL747A NEL747B Kit TSA DNP (AP chromogen) TSA DNP (AP chromogen) TSA DNP (HRP chromogen) TSA DNP (HRP chromogen) Solvent DMSO DMSO DMSO DMSO Reconstitution volume 0.3 mL 0.15 mL 0.3 mL 0.15 mL 1 DNP Amplification Reagent Working Solution Before each procedure, dilute the DNP Stock Solution 1:50 using 1X Plus Amplification Diluent to make the DNP Amplification Reagent Working Solution. Approximately 100-300 µL of DNP Amplification Reagent Working Solution is required per slide. Discard any unused portion of working solution. Reagent Titration In general, most researchers have found that TSA requires lower probe and conjugate concentrations for optimal results when compared with standard unamplified nonradioactive methods. 1. Probe titration Probe concentration must be optimized. It should be assessed using the standard concentration used in unamplified nonradiometric procedures, and at 1:2 to 1:20 dilutions in hybridization mix. Failure to establish appropriate probe concentration can result in little to no signal development. 2. Titration of HRP enzyme conjugate in TNB Blocking Buffer HRP must be present in the system. This can be accomplished using haptenlabeled probes followed by an appropriate anti-hapten-HRP conjugate. Streptavidin-HRP is supplied with the kit. Other HRP conjugates can be purchased separately. Appropriate HRP conjugate concentrations to assess include supplier’s recommended starting concentration, 1:2 dilution, and 1:5 dilution. HRP dilutions should be done in TNB Blocking Buffer. In cases where no signal and no background are seen, it may be necessary to increase concentration instead. Quenching Endogenous Peroxidase Amplification for all DNP kits is catalyzed by HRP. To minimize background, endogenous peroxidase activity must be quenched. Users should establish the need for doing this and optimal methodology specific to the tissues or cells being stained. For paraffin-embedded tissues, quenching can be done after dewaxing and alcohol rehydration but before the protease digestion step. After quenching wash with TNT or 1X PBS wash buffer for 5 minutes. Recommended quenching solution: 0.3% - 3% H2O2 in PBS or methanol, 10-60 minute incubation Volumes The protocol is written for minimal volumes of reagent (e.g., 100-300 µL). Reagent volumes used should be sufficient to completely cover cells or tissue sections on slide. 2 TSA DNP Systems Immunohistochemistry (IHC) Protocol 1. Prepare tissues or cells using standard fixation and embedding techniques. Dewax and rehydrate slides according to standard procedures. 2. Follow standard non-radioactive in situ hybridization techniques. Include tissue permeabilization (if needed) and quenching of endogenous peroxidase activity (if needed, see p. 2). Probe hybridization (with digoxigenin, biotin, or fluoresceinlabeled probes) should be done using concentration determined in optimization studies (see Reagent Titration, p. 2) followed by posthybridization stringency washes. NOTE: Always run an unamplified control slide and an amplified negative control slide with each experiment. Blocking Step 3. Incubate slides with 100-300 µL of TNB Blocking Buffer in a humidified chamber for 30 minutes at room temperature. Introduction of HRP (both the TSA DNP AP and TSA DNP HRP kits require an HRP for this amplification step) 4. Drain off TNB Blocking Buffer. Add 100-300 µL of your HRP (diluted in TNB Blocking Buffer) to each slide and place a coverslip on top to reduce evaporation. Incubate the slides in a humid chamber at room temperature for 30 minutes with agitation. a) DIG-labeled probes: 100-300 µL of antidigoxigenin-HRP (Roche Applied Science anti-DIG-POD Cat. #11207733910) diluted 1:100 in TNB Buffer. b) Biotin-labeled probes: 100-300 µL of streptavidin-HRP (Cat. # NEL750 or supplied in kit) diluted 1:2000 in TNB Buffer. c) Fluorescein-labeled probes: 100-300 µL of anti-fluorescein-HRP (Cat. # NEF710) diluted 1:250 in TNB Buffer.. 5. Wash slides three times (5 minutes per wash) in fresh TNT Wash Buffer at room temperature. Amplification 6. Pipet 100-300 µL of your DNP Amplification Reagent Working Solution onto each slide. Incubate the slides at room temperature for 3 to 10 minutes. 7. Wash the slides three times (5 minutes per wash) in TNT Wash Buffer at room temperature with agitation. 3 Visualization 8. Follow desired chromogenic visualization option: a) AP-Chromogenic Option (kits NEL746A/NEL746B) • • • • Add 100-300 µL of anti-DNP-AP (provided in kit) diluted 1:100 in TNB Buffer to each slide. Incubate the slides in a humidified chamber at room temperature for 30 minutes. Wash the slides 3X for 5 minutes each in TNT Buffer at room temperature with agitation. Visualize with 100-300 µL standard alkaline phosphatase chromogenic substrates such as BCIP/NBT (5-bromo-4-chloro-indolyl phosphate/nitroblue tetrazolium). Incubate slides ten minutes in the dark. Examine slides for signal strength. If darker signal is desired, incubate slides for up to an additional 10-30 minutes. Counterstain if desired. Nuclear Fast Red is an effective counterstain for BCIP/NBT. Histomount™ and Clearmount™ may be used for mounting. b) HRP-Chromogenic Option (kits NEL747A/NEL747B) • • • • 100-300 µL of anti-DNP-HRP (provided in kit) diluted 1:100 in TNB Buffer to each slide. Incubate the slides in a humidified chamber at room temperature for 30 minutes. Wash the slides 3X for 5 minutes each in TNT Buffer at room temperature with agitation. Visualize with standard HRP chromogenic substrates such as DAB (diaminobenzidine) or AEC(aminoethyl carbazole). Incubate 5 minutes in the dark. Counterstain if desired. Hematoxylin is an effective counterstain for DAB and AEC. Histomount™ and Clearmount™ may be used for mounting DAB-stained slides. Use aqueous mounting media with AEC. 4