5-Formylcytosine (5-fC) (D5D4K) Rabbit mAb #74178

Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

Stock Solutions

• 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.
• Ethanol, 70% solution, deionized.
• 1.5 M Hydrochloric acid.

• 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 9.0 mL dH₂O, mix well. 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 and 0.5 mL 20X PBS to 9.5 mL dH₂O. Mix well then add 0.1 g BSA (#9998), mix.

• Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:

  • Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 488 Conjugate) #4412
  • Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 555 Conjugate) #4413
  • Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 594 Conjugate) #8889
  • Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 647 Conjugate) #4414
• Prolong^® Gold AntiFade Reagent (#9071), Prolong^® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Cultured Cell Lines (IF-IC)

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

1. Aspirate media, cover cells completely with ice-cold 70% ethanol.
2. Allow cells to fix for 5 minutes at room temperature.
3. Aspirate fixative, rinse three times in 1X PBS for 5 minutes each.
4. Add 1.5 M HCl and incubate for 30 minutes at room temperature.
5. Aspirate HCl and rinse two times in 1X PBS for 5 minutes each.
6. 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 minutes.
2. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
3. Aspirate blocking solution, apply diluted primary antibody.
4. Incubate overnight at 4°C.
5. Rinse three times in 1X PBS for 5 minutes each.
6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hours at room temperature in dark.
7. Rinse three times in 1X PBS for 5 minutes each.
8. Mount samples in an appropriate antifade reagent such as 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.

DNA Dot Blot Protocol

A. Buffers and Reagents

1. 20X Saline Sodium Citrate (SSC) Buffer: 3.0 M NaCl, 0.3 M Sodium Citrate, pH to 7.0.
2. 10X SSC Buffer: Dilute 20X SSC buffer 1:2.
3. 2X DNA Denaturing Buffer: 200 mM NaOH, 20 mM EDTA.
4. Nuclease-Free Water: (#12931)
5. Blotting Membrane: This protocol has been optimized for positively charged nylon membranes.
6. 96-Well Dot Blot Apparatus
7. 10X Tris Buffered Saline with Tween^® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2 0, mix.
8. Nonfat Dry Milk: (#9999)
9. Blocking Buffer: 1x TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.
10. Bovine Serum Albumin (BSA): (#9998)
11. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well.
12. Secondary Antibody Conjugated to HRP: anti-rabbit (#7074); anti-mouse (#7076).
13. Detection Reagent LumiGLO^® chemiluminescent reagent and peroxide (#7003) or SignalFire™ ECL Reagent (#6883)

B. Dot Blot

Note: This protocol is written for spotting fragmented, purified genomic DNA (titration of 1000 ng, 500 ng, 250 ng, 125 ng, 62.5 ng, 31.25 ng, and 15.625 ng) onto a positively charged nylon membrane using a 96-well dot blotting apparatus. Depending on the source and type of DNA, more or less DNA may be required for detection with the antibody.
Before Starting:
• Purify genomic DNA using a genomic DNA purification protocol or kit and sonicate
genomic DNA to generate fragments between 200 and 500 bp. DNA fragment size
can be analyzed by gel electrophoresis on a 1% agarose gel with a 100 bp DNA
marker.
• Cut a piece of nylon membrane to the size of the dot blot manifold.
• Wet nylon membrane with 10x SSC Buffer.
• Dry membrane by placing it in 96-well dot blot apparatus and applying vacuum.

1. Dilute fragmented genomic DNA to 100 ng/μl in 100 ul of nuclease-free water.
   Then denature DNA by adding 100 μl of 2X DNA Denaturing Buffer and incubating at 95°C for 10 min.
2. Add 200 μl of 20X SSC buffer and immediately chill on ice for 5 min.
3. Add 100 μl of nuclease-free water to bring DNA solution to a final volume of 500 μl with a DNA concentration of 20 ng/μl.
4. Set up a series of six 2-fold dilutions by adding 250 μl of the DNA solution, starting with the DNA solution in Step 3, to 250 μl of nuclease-free water. This will generate seven DNA samples containing 250 μl DNA at concentrations of 20 ng/μl, 10 ng/μl, 5 ng/μl, 2.5 ng/μl, 1.25 ng/μl, 0.625 ng/μl, and 0.3125 ng/μl.
5. Apply 50 μl of each of the seven dilution samples into separate wells of the 96-well dot blot apparatus, leaving the last well for nuclease-free water only. The amount of DNA added to each well should then be 1000 ng, 500 ng, 250 ng, 125 ng, 62.5 ng, 31.25 ng, 15.625 ng and 0 ng respectively. Apply gentle vacuum pressure to draw solution through the membrane. Nylon membrane should be mostly dry before step 6.
6. Remove nylon membrane from the 96-well dot blot apparatus and wrap in plastic wrap.
7. UV cross-link nylon membrane at 1200 J/m².

C. Membrane Blocking and Antibody Incubation

1. Incubate membrane in 25 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
2. Wash membrane three times for 5 min each with 15 ml of 1X TBST.
3. Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the antibody product datasheet) in 10 ml primary antibody dilution buffer, with gentle agitation overnight at 4°C.
4. Wash three times for 5 min each with 15 ml of 1X TBST.
5. Incubate membrane with the species appropriate HRP-conjugated secondary antibody (#7074 Anti-rabbit IgG, HRP-linked Antibody or #7076 Anti-mouse IgG, HRP-linked Antibody) at 1:2000 in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
6. Wash membrane three times for 5 min each with 15 ml of 1X TBST.
7. Proceed with detection (Section D)

D. Detection of DNA

1. Incubate membrane with 10 mL of LumiGLO^® (0.5 ml 20x LumiGLO^® #7003, 0.5 ml 20x Peroxide, and 9.0 ml purified water) or 10 ml SignalFire™ #6883 (5 ml Reagent A, 5 ml Reagent B) with gentle agitation for 1 min at room temperature.
2. Drain membrane of excess developing solution (do not let dry), wrap in plastic wrap and expose to x-ray film. An initial 10 sec exposure should indicate the proper exposure time.

NOTE: Due to the kinetics of the detection reaction, signal is most intense immediately following incubation and declines over the following 2 hr