Western Blotting (WB), Immunofluorescence (IF), Immunocytochemistry (ICC)
Specificity
Immunohistochemistry-Paraffin Embedded Sections Note: SEMA3B protein is secreted and accumulates in the endoplasmic reticulum. Antigen Unmasking: Bring slides to a boil in 10 mM sodium citrate buffer (pH 6.0) then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench-top for 30 minutes. Staining: 1. Wash sections in deionized water three times for 5 minutes each. 2. Wash sections in wash buffer for 5 minutes. 3. Block each section with 100-400 µL blocking solution for 1 hour at room temperature. 4. Remove blocking solution and add 100-400 µL diluted primary antibody. Incubate overnight at 4 C. 5. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each. 6. Add 100-400 µL biotinylated diluted secondary antibody. Incubate 30 minutes at room temperature. 7. Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each. 8. Add 100-400 µL Streptavidin-HRP reagent to each section and incubate for 30 minutes at room temperature. 9. Wash sections three times in wash buffer for 5 minutes each. 10. Add 100-400 µL DAB substrate to each section and monitor staining closely. 11. As soon as the sections develop, immerse slides in deionized water. 12. Counterstain sections in hematoxylin. 13. Wash sections in deionized water two times for 5 minutes each. 14. Dehydrate sections. 15. Mount coverslips.
Purification
Aff - Purified
Immunogen
A synthetic peptide made to an internal portion of the human SEMA 3B protein sequence (AA100-200).
SEMA3B
Reactivity: Human
WB
Host: Rabbit
Polyclonal
unconjugated
Application Notes
Western Blot 1:5000 , Immunohistochemistry: 1:100 (paraffin-embedded tissue), Immunocytochemistry/Immunofluorescence 1:40 Dilutions listed as a recommendation. Optimal dilution should be determined by investigator.
Restrictions
For Research Use only
Format
Liquid
Concentration
1.0 mg/mL
Buffer
Tris-glycine, 150 mM NaCL and 0.05% Sodium Azide as a preservative.
Preservative
Sodium azide
Precaution of Use
WARNING: Reagents contain sodium azide. Sodium azide is very toxic if ingested or inhaled. Avoid contact with skin, eyes, or clothing. Wear eye or face protection when handling. If skin or eye contact occurs, wash with copious amounts of water. If ingested or inhaled, contact a physician immediately. Sodium azide yields toxic hydrazoic acid under acidic conditions. Dilute azide-containing compounds in running water before discarding to avoid accumulation of potentially explosive deposits in lead or copper plumbing.
Handling Advice
Repeated freeze/thaw cycles
Storage
-20 °C
Storage Comment
Store frozen. Aliquot as undiluted antisera and immediately place at -20°C. Antisera may have become trapped in top of vial during shipping. Centrifugation of vial is recommended before opening. Stable for at least 6 months at -20°C. Compromise the integrity of the antiserum.