CST Guide:

Section II: ANTIBODY APPLICATIONS
Cultured Cells (Immunocytochemistry, IF-IC) Protocol
IMPORTANT: Please refer to the Applications section on the front page of the product’s datasheet or product webpage to
determine if this product is validated and approved for use on cultured cell lines (IF-IC), frozen tissue sections (IF-F), or paraffinembedded
samples (IF-P). Some of CST’s antibodies perform optimally using an alternate protocol. Please consult the datasheet
or webpage for product-specific recommendations.
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH 2 O, mix.
NOTE: Adjust pH to 8.0.
2. Formaldehyde, 16%, methanol free: (Polysciences, Inc., cat #18814), use fresh, store opened vials at 4°C in dark.
Dilute 1 in 4 in 1X PBS to make a 4% formaldehyde solution.
3. Blocking Buffer: (1X PBS/5% normal serum/0.3% Triton X-100): To prepare 10 ml, add 0.5 ml normal serum from the
same species as the secondary antibody (e.g., Normal Goat Serum (#5425) and 0.5 ml 20X PBS to 9 ml dH 2 O) and mix
well. While stirring, add 30 µl Triton X-100.
4. 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 0.1g BSA (#9998), mix until well dissolved.
5. Fluorochrome-conjugated Secondary Antibodies: (Anti-mouse #4408, #4409, #8890, #4410) (Anti-rabbit #4412, #4413,
#8889, #4414) (Anti-rat #4416, #4417, #4418)
6. Mounting Medium: ProLong ® Gold Antifade Reagent (#9071) or ProLong ® Gold Antifade Reagent with DAPI (#8961)
B. Specimen Preparation
I. Cultured Cell Lines (IF-IC)
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.
2. Allow cells to fix for 15 min at room temperature.
3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
4. Proceed with Immunostaining (Section C).
C. Immunostaining
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.
1. Block specimen in blocking buffer for 60 min.
2. While blocking, prepare primary antibody by diluting as indicated on the product’s datasheet or webpage in antibody dilution
buffer.
3. Aspirate blocking solution, apply diluted primary antibody.
4. Incubate overnight at 4°C.
5. Rinse 3 times in 1X PBS for 5 min each. NOTE: If using a fluorochrome-conjugated primary antibody, proceed directly to
Step 8.
6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer for 1–2 hr at room
temperature in the dark.
7. Rinse 3 times in 1X PBS for 5 min each.
8. Coverslip slides with ProLong ® Gold Antifade Reagent (#9071) or ProLong ® Gold Antifade Reagent with DAPI (#8961).
9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C
protected from light.
Frozen/Cryostat Tissue Sections (IF-F) Protocol
IMPORTANT: Please refer to the Applications section on the front page of the product’s datasheet to determine whether an
antibody is validated and approved for use on cultured cell lines (IF-IC), frozen tissue sections (IF-F), or paraffin-embedded
samples (IF-P). Some of CST’s antibodies perform optimally using an alternate protocol. Please consult the datasheet or webpage
for product-specific recommendations.
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS, add 50 ml 20X PBS to 950 ml dH 2 O, mix.
NOTE: Adjust pH to 8.0.
2. Formaldehyde: 16%, methanol free: (Polysciences, Inc., cat #18814) Use fresh and store opened vials at 4°C in dark.
Dilute in 1X PBS to make a 4% formaldehyde solution.
3. Blocking Buffer (1X PBS/5% normal serum/0.3% Triton X-100): To prepare 10 ml, add 0.5 ml normal serum from
the same species as the secondary antibody (e.g., Normal Goat Serum (#5425) and 0.5 ml 20X PBS to 9 ml dH 2 O and mix
well. While stirring, add 30 µL Triton X-100.
4. 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. Add 0.1 g BSA (#9998) and mix well until dissolved.
5. Fluorochrome-conjugated Secondary Antibodies: Anti-mouse (#4408, #4409, #8890, #4410), Anti-rabbit (#4412,
#4413, #8889, #4414), Anti-rat (#4416, #4417, #4418)
6. Mounting Medium: ProLong ® Gold Antifade Reagent (#9071) or ProLong ® Gold Antifade Reagent with DAPI (#8961)
B. Specimen Preparation
1. For fixed frozen tissue, allow sections to air dry on the bench for 10 min, then proceed with Immunostaining (Section C).
2. For fresh, unfixed frozen tissue, fix immediately as follows:
a. Cover sections to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS. NOTE: Formaldehyde is toxic, use only
in a fume hood.
b. Allow sections to fix for 15 min at room temperature.
c. Aspirate liquid, rinse slides 3 times in 1X PBS for 5 min each.
d. Proceed with Immunostaining (Section C).
C. Immunostaining
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.
1. Block specimen in Blocking Buffer for 60 min.
2. While blocking, prepare primary antibody by diluting as indicated on the product’s datasheet or webpage in antibody dilution
buffer.
3. Aspirate Blocking Buffer, then apply diluted primary antibody.
4. Incubate overnight at 4°C.
5. Rinse 3 times in 1X PBS for 5 min each. NOTE: If using a fluorochrome-conjugated primary antibody, proceed directly to
Step 8.
6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer for 1 hr in the dark.
7. Rinse 3 times in 1X PBS for 5 min each.
8. Mount slides with ProLong ® Gold Antifade Reagent (#9071) or ProLong ® Gold Antifade Reagent with DAPI (#8961).
9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C
protected from light.
chapter 11: Immunofluorescence (IF)
Phalloidin labels actin filaments
and DRAQ5 ® labels DNA.
Alexa Fluor ® 555 Phalloidin #8953: Confocal IF analysis of HeLa cells using COX IV
(3E11) Rabbit mAb (Alexa Fluor ® 488 Conjugate) #4853 (green). Actin filaments were
labeled with #8953 (red). Blue pseudocolor = DRAQ5 ® #4084 (fluorescent DNA dye).
ProLong ® Gold Antifade Reagent
extends fluorescent signal, allowing
more time to capture images.
ProLong ® Gold Antifade Reagent #9071: Confocal IF analysis of SNB19 cells using
β–Actin (8H10D10) Mouse mAb #3700 (red), Vimentin (D21H3) XP ® Rabbit mAb (Alexa
Fluor ® 488 Conjugate) #9854 (green), and COX IV (3E11) Rabbit mAb (Alexa Fluor ® 647
Conjugate) #7561 (blue).
210 For Research Use Only. Not For Use in Diagnostic Procedures. See pages 302 & 303 for Pathway Diagrams, Application, and Reactivity keys.
www.cellsignal.com/cstprotocols
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