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Pan-Keratin (C11) Mouse mAb (Alexa Fluor® 488 Conjugate) #4523
Confocal immunofluorescent analysis of paraffin-embedded human breast carcinoma using Pan-Keratin (C11) Mouse mAb (Alexa Fluor® 488 Conjugate) (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).Learn more about how we get our images
Gallery: Pan-Keratin (C11) Mouse mAb (Alexa Fluor® 488 Conjugate) #4523
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
- Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
- Blocking Buffer (1X PBS / 5% normal goat serum (#5425) / 0.3% Triton™ X-100): To prepare 10 ml: add 0.5 ml normal goat serum and 0.5 ml 20X PBS to 9.0 ml dH2O, mix. While stirring, add 30 µl Triton™ X-100.
- Antibody Dilution Buffer: (1X PBS / 1% BSA / 0.3% Triton™ X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (#9998), mix.
- Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).
Reagents specific to IF-P application:
- Ethanol, anhydrous denatured, histological grade, 100% and 95%.
- Antigen Unmasking:
- For Citrate: 10 mM Sodium Citrate Buffer: To prepare 1 L add 2.94 g (C6H5Na3O7•2H2O) to 1 L dH2O. Adjust pH to 6.0.
- For EDTA: 1 mM EDTA: To prepare 1 L add 0.372 g EDTA (C10H14N2O8Na2•2H2O) to 1 L dH2O. Adjust pH to 8.0.
B. Specimen Preparation - Paraffin Sections (IF-P)
NOTE: Do not allow slides to dry at any time during this process.
- Wash three times in xylene for 5 min each.
- Wash two times in 100% ethanol for 10 min each.
- Wash two times in 95% ethanol for 10 min each.
- Rinse sections two times in dH2O for 5 min each.
- Antigen Unmasking: NOTE: Consult product datasheet or product webpage for specific recommendation for the unmasking solution.
- For Citrate: Bring slides to a boil in 10 mM sodium citrate buffer pH 6.0, then maintain at a sub-boiling temperature for 10 min. Cool slides on bench top for 30 min.
- For EDTA: Bring slides to a boil in 1 mM EDTA pH 8.0 followed by 15 min at a sub-boiling temperature. No cooling is necessary.
- Proceed with Immunostaining (Section C).
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
- Block specimen in Blocking Buffer for 60 min.
- While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
- Aspirate blocking solution, apply diluted primary antibody.
- Incubate overnight at 4°C.
- Rinse three times in 1X PBS for 5 min each.
- Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
- For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.
posted November 2006
revised November 2013
protocol id: 201