CST Guide:

Section II: ANTIBODY APPLICATIONS
Paraffin Tissue Sections (IF-P) 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 or equivalent grade water (dH 2 O).
1. Xylene
2. Ethanol, anhydrous denatured, histological grade (100% and 95%)
3. Antigen Unmasking:
a. 10 mM Sodium Citrate Buffer: To prepare 1 L add 2.94 g sodium citrate trisodium salt dihydrate (C 6 H 5 Na 3 O 7 •2H 2 O)
to 1 L dH 2 O. Adjust pH to 6.0.
b. 1 mM EDTA: To prepare 1 L add 0.372 g EDTA (C 10 H 14 N 2 O 8 Na 2 • 2 H 2 O) to 1 L dH 2 O. Adjust pH to 8.0.
4. 10X Tris Buffered Saline with Tween ® 20 (TBST): (#9997) To prepare 1 L 1X TBST, add 100 ml 10X TBST to 900 ml
dH 2 O, mix.
5. Incubation Buffer (1X TBST/5% normal serum): To prepare 10 ml, add 0.5 ml normal serum from the same species as
the secondary antibody (e.g., Normal Goat Serum (#5425) to 9.5 ml of 1X TBST, mix well.
6. Fluorochrome-conjugated Secondary Antibodies: Anti-mouse (#4408, #4409, #8890, #4410), Anti-rabbit (#4412,
#4413, #8889, #4414), Anti-rat (#4416, #4417, #4418)
7. Mounting Medium: ProLong ® Gold Antifade Reagent (#9071) or ProLong ® Gold Antifade Reagent with DAPI (#8961)
B. Deparaffinization/Rehydration
NOTE: Do not allow slides to dry at any time during this procedure.
1. Incubate sections in 3 washes of xylene for 5 min each.
2. Incubate sections in 2 washes of 100% ethanol for 10 min each.
3. Incubate sections in 2 washes of 95% ethanol for 10 min each.
4. Wash sections 2 times in dH 2 O for 5 min each.
C. Antigen Unmasking
NOTE: Consult product datasheet for specific recommendation for the unmasking solution/protocol.
1. For Citrate: Bring slides to a boil in 10 mM Sodium Citrate Buffer, pH 6.0; maintain at a sub-boiling temperature (95–98°C)
for 10 min. Cool slides on bench top for 30 min.
2. For EDTA: Bring slides to a boil in 1 mM EDTA, pH 8.0. Follow with 15 min at a sub-boiling temperature (95–98°C). No
cooling is necessary.
D. 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. Wash sections 3 times in 1X TBST for 5 min each.
2. Block specimen in Incubation Buffer for 60 min.
3. While blocking, prepare primary antibody by diluting as indicated on the product’s datasheet or webpage in Incubation Buffer.
4. Aspirate buffer from sections; apply diluted primary antibody.
5. Incubate overnight at 4°C.
6. Wash 3 times in 1X TBST for 5 min each. NOTE: If using a fluorochrome-conjugated primary antibody, proceed directly to
Step 9.
7. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Incubation Buffer for 1 hr at room temperature
in the dark.
8. Rinse 3 times in 1X TBST for 5 min each.
9. Mount slides with ProLong ® Gold Antifade Reagent (#9071) or ProLong ® Gold Antifade Reagent with DAPI (#8961).
10. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C
protected from light.
In-Cell Western Protocol
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis or equivalently purified water (dH 2 O).
1. 10X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS add 80 g sodium chloride (NaCl), 2 g potassium chloride
(KCl), 14.4 g sodium phosphate, dibasic (Na 2 HPO 4 ) and 2.4 g potassium phosphate, monobasic (KH 2 PO 4 ) to 1 L dH 2 O. Adjust
pH to 7.4.
2. Formaldehyde, 16%, methanol-free: (Polysciences, Inc. #18814) Use fresh; store opened vials at 4°C in dark, dilute in
1X PBS for use.
3. Blocking Buffer: To prepare 25 ml, add 2.5 ml 10X PBS, 1.25 ml normal serum from the same species as the secondary
antibody (e.g., normal goat serum (#5425), normal donkey serum) and 21.25 ml dH 2 O and mix well. While stirring, add 75 µl
Triton X-100 (100%).
4. Antibody Dilution Buffer: To prepare 40 ml, add 4 ml 10X PBS to 36 ml dH 2 O, mix. Add 0.4 g BSA and mix well. While
stirring, add 120 µl Triton X-100 (100%).
5. Fluorochrome-conjugated secondary antibody.
NOTE: When using any primary or fluorochrome-conjugated secondary antibody for the first time, titrate the antibody to
determine which dilution allows for the strongest specific signal with the least background for your sample.
B. Fixation and Immunolabeling
NOTE: Please refer to the IF protocol for each antibody to identify the recommended antibody-specific fixation and permeabilization
conditions. Cells should be grown, treated, fixed, and stained directly in multi-well plates. To avoid edge effects from uneven
distribution of cells in individual wells, let newly-seeded plates stand at room temperature for one hour before placing in 37°C
incubator (for more information, see Lundholt, B.K., Scudder, K.M., and Pagliaro, L. (2003) A simple technique for reducing
edge effect in cell-based assays. J. Biomol. Screen. 8, 566–570). To minimize false signal from scattered light and background
fluorescence, use black-walled multi-well plates (e.g., BD Falcon, #353948). Avoid touching the inside of the wells with pipettes
or aspirators as this may leave an artifact that will affect subsequent quantification. Instead, empty wells by shaking inverted
plate sharply over waste receptacle and then blotting with clean paper towel. Volumes indicated below are for use with standard
96-well plates. Adjust volume accordingly for plates with larger or smaller wells.
1. Grow and treat cells as desired in multi-well plates.
2. Fix cells with 50 µl 4% formaldehyde or 100% methanol, as recommended in the antibody-specific IF protocol.
3. Allow cells to fix for 15 min at room temperature.
4. Shake inverted plate over aldehyde waste receptacle and blot with clean paper towel.
5. Rinse plate 3 times for 5 min each with room temperature PBS (100 µl/well).
6. Block cells in Blocking Buffer for one hour at room temperature (50 µl/well).
7. While blocking, prepare primary antibody by diluting in Antibody Dilution Buffer as indicated for Immunofluorescence (IF-IC)
on datasheet.
8. Shake out blocking solution and add diluted primary antibody (total volume 50 µl/well).
NOTE: For double-labeling, prepare a cocktail of mouse and rabbit primary antibodies at their appropriate dilutions in
Antibody Dilution Buffer.
9. Cover plate and incubate overnight at 4°C.
10. Rinse plate 3 times in PBS for 5 min each.
11. Add fluorochrome-conjugated secondary antibody* diluted in Antibody Dilution Buffer (total volume 50 µl/well) and incubate
for 1 hr at room temperature in the dark.
NOTE: For double-labeling, prepare a cocktail of fluorochrome-conjugated anti-mouse and anti-rabbit secondary antibodies
in Antibody Dilution Buffer.
12. Rinse plate 3 times in PBS for 5 min each.
13. To normalize for cell number, label nuclei using DRAQ5 ® diluted 1:1000 in PBS (total volume 50 µl/well). Incubate at least
30 min at room temperature in the dark prior to scanning.
NOTE: DRAQ5 ® can be emptied and the wells rinsed at least 1X with PBS for better optical resolution.
14. Scan plate according to manufacturer’s directions. (Be sure bottom of plate has been wiped clean prior to scanning).
Recommended Secondary Antibodies: Anti-Rabbit: Anti-Rabbit IgG (H+L) (DyLight ® 680 Conjugate) #5366, Anti-Rabbit IgG
(H+L) (DyLight ® 800 Conjugate) #5151, Anti-Mouse: Anti-Mouse IgG (H+L) (DyLight ® 680 Conjugate) #5470, Anti-Mouse IgG
(H+L) (DyLight ® 800 Conjugate) #5257
chapter 11: Immunofluorescence (IF)
IF Companion Products
See our comprehensive list of IF companion products that are validated in-house with our protocols so that you can
get the reliable reagents you need to complete your experiment. www.cellsignal.com/ifcompanion
212 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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