5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb #51660

Immunofluorescence (Immunocytochemistry)

IMPORTANT: This protocol employs an atypical fixation and denaturation strategy with which only certain targets are compatible. Where appropriate, this protocol will be linked to its validated antibody under the Product Information banner on the product-specific webpage.

A. Solutions and Reagents

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

1. 1X Phosphate Buffered Saline (PBS): To prepare 1L 1X PBS, add 100 ml 10X Wash Buffer, Phosphate Buffered Saline (#12528) to 900 ml dH₂O, mix. Adjust pH to 8.0.
2. Ethanol , 70% solution, deionized.
3. 1.5 M Hydrochloric acid.
4. Blocking Buffer: Purchase ready-to-use Immunofluorescence Blocking Buffer (#12411), or prepare a 1X PBS / 5% normal serum / 0.3% Triton^™ X-100 buffer by adding 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425)) and 30 µl Triton^™ X-100 to 9.5 mL 1X PBS. Store at 4°C.
5. Antibody Dilution Buffer: Purchase ready-to-use Immunofluorescence Antibody Dilution Buffer (#12378), or prepare a 1X PBS / 1% BSA / 0.3% Triton^™ X-100 buffer by adding 0.1 g BSA (#9998) and 30 µl Triton^™ X-100 to 10 mL 1X PBS. Store at 4°C.
6. Fluorochrome-conjugated Secondary Antibody : Use a secondary antibody that is reactive to your primary antibody host species (e.g., rabbit). Click here for a list of secondary antibodies approved for immunofluorescence.

B. Fixation and Permeabilization

NOTE: All subsequent incubations should be carried out at room temperature (20-25°C) unless noted otherwise.

1. Aspirate media then cover specimen with ice-cold 70% ethanol (use enough to cover completely to a depth of 3 - 5 mm, DO NOT LET DRY).
2. Allow to fix for 5 min.
3. Rinse three times in 1X PBS for 5 min each.
4. Aspirate PBS then incubate fixed specimen in 1.5 M HCl for 1 hour.
5. Rinse two times in 1X PBS for 5 min each.

C. Immunostaining

1. Block specimen in Blocking Buffer for 60 min.
2. While blocking, prepare primary antibody in Antibody Dilution Buffer (see product website for recommended dilution range).
3. Aspirate blocking solution then apply diluted primary antibody.
4. Incubate overnight at 4°C.

5. Rinse three times in 1X PBS for 5 min each.

NOTE: If using a fluorochrome-conjugated primary antibody, then skip to Section C, Step 8.

6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hours protected from light.
7. Rinse three times in 1X PBS for 5 min each protected from light.

8. Counterstain as appropriate.

NOTE: When including fluorescent cellular dyes in your experiment (DNA dyes, etc.), please refer to the dye product page for its recommended protocol. View our listing of cellular dyes validated for use in immunofluorescence.

9. Mount specimen in an appropriate antifade reagent such as Prolong^® Gold Antifade Reagent (#9071) or Prolong^® Gold AntiFade Reagent with DAPI (#8961).
10. For long-term storage, store samples 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