Table 1. Solution recipes.
Composition for the solutions used in this protocol in order of appearance. All the stocks are liquids unless otherwise noted. The chemicals were resuspended or diluted in autoclaved filtered water unless otherwise noted.
| RECIPES (in order of appearance in protocol) | ||
|---|---|---|
| All stocks are liquids unless otherwise noted | ||
| Chemicals are resuspended or diluted in autoclaved filtered water unless otherwise noted | ||
| 50% bleach | ||
| 1 part standard (i.e., non-concentrated) commercial bleach (6% sodium hypochlorite) | ||
| 1 part tap water | ||
| Fixation buffer | stock | comments |
| 4% paraformaldehyde | 16% | stock stored in small glass ampules |
| 1× phosphate-buffered saline | 10× | |
| Blocking solution | stock | comments |
| 1× phosphate-buffered saline | 10× | |
| 10% bovine serum albumin | 30% | |
| 0.1% Tween-20 | 10% (v/v) | stock diluted from original 100% liquid |
| 0.1% sodium azide | 1% (w/v) | stock prepared from powder; TOXIC |
| Antibody solution | stock | comments |
| 1× phosphate-buffered saline | 10× | |
| 5% bovine serum albumin | 30% | |
| 0.1% Tween-20 | 10% (v/v) | stock diluted from original 100% liquid |
| 0.1% sodium azide | 1% (w/v) | stock prepared from powder; TOXIC |
| PBS-Tween | stock | comments |
| 1× phosphate-buffered saline | 10× | |
| 1% bovine serum albumin | 30% | |
| 0.1% Tween-20 | 10% (v/v) | stock diluted from original 100% liquid |
| PDMS | ||
| 10 parts Sylgard silicone elastomer base | ||
| 1 part Sylgard elastomer curing agent | ||
| Activation solution | stock | comments |
| 1 mM MA-NHS* | 1 M | stock resuspended in DMSO |
| 1× phosphate-buffered saline | 10× | |
| * methylacrylic acid N-hydroxysuccinimidyl ester | ||
| Monomer solution* | stock | comments |
| 2 M NaCl | 5 M | |
| 1× phosphate-buffered saline | 10× | |
| 8.625% (w/v) sodium acrylate | powder | TOXIC |
| 2.5% (w/v) acrylamide | powder | TOXIC |
| 0.15% (w/v) bisacrylamide | powder | TOXIC |
| *Monomer solution can be stored at 4°C for up to two weeks | ||
| Gelation solution | stock | comments |
| Add ingredients in this order: | ||
| 3920 μl monomer solution (above) | ||
| 60 μl TEMED* | 10% (v/v) | diluted in H2O; keep stock at -20°C |
| 20 μl TEMPO** | 1% (w/v) | diluted in H2O; prepare fresh from powder |
| Add last: | ||
| 80 μl ammonium persulfate | 10% (w/v) | diluted in H2O; keep stock at -20°C |
| *N,N,N',N'-tetramethylethane-1,2-diamine | ||
| ** (2,2,6,6-tetramethylpiperidin-1-yl)oxyl | ||
| Digestion buffer | stock | comments |
| 1× TAE | 10× | |
| 80 mM guanidine HCl | powder | TOXIC |
| 0.1% Tween-20 | 10% (v/v) | stock diluted from original 100% liquid |
| Add last: | ||
| 250 μl proteinase K ( 20 mg/ml, 600 U/ml) to 20 ml digestion buffer |