Protocol

This is the current, August 2026 version of the REPLICA protocol. For any queries, please contact James Manton via jmanton@mrclmb.ac.uk.

In brief, we first fix and wash the sample with detergent to facilitate antibody penetration. Primary antibodies are used as supplied, but secondary antibodies must first be functionalised with vinyl groups using, for example, acryloyl-X in order to ensure their incorporation into the hydrogel. After staining, the tissue is infused with a monomer solution inhibited from polymerisation before a new monomer solution with initiation and accelerant is added. A series of enzymatic incubations then digests the sample, with fragments washed out using SDS. Finally, the sample is washed using water, which also acts as the refractive index matching liquid.

Preincubation monomer solution

All items are purchased from Sigma-Aldrich.

Component Cat. no. Stock w/w % Final w/w % μl stock per ml final
Acrylamide A4058 40 7.5 187.5
Bisacrylamide M1533 2 0.25 125
4-hydroxy-TEMPO 176141 1 0.001 1
Water — — — 686.5

Polymerisation monomer solution

All items are purchased from Sigma-Aldrich.

Component Cat. no. Stock w/w % Final w/w % μl stock per ml final
Acrylamide A4058 40 7.5 187.5
Bisacrylamide M1533 2 0.25 125
4-hydroxy-TEMPO 176141 1 0.001 1
TEMED T9281 10 0.15 15
APS A3678 10 0.15 15
Water — — — 656.5

Buffers

Blocking buffer: 4 % normal donkey serum, 0.25 % Triton X-100 in PBS

Enzyme digestion buffer: 10 mM Tris-HCl, 2.5 mM MgCl₂, 0.5 mM CaCl₂ @ pH 7.6

Cleaning buffer: 200 mM SDS, 200 mM NaCl, 50 mM HEPES @ pH 7.0

Antibody functionalisation

Antibodies must be functionalised with vinyl groups to facilitate incorporation into the hydrogel network during polymerisation. For this we typically use acryloyl-X (AcX), although other similar reagents should also work well. Normally, only secondary antibodies are functionalised.

Prior to functionalisation, AcX powder should be dissolved in anhydrous DMSO to a concentration of 2 mg / ml. If antibodies are stored with sodium azide, this must be removed first, e.g. using a ZebaSpin desalting column.

  1. Add 200 mM sodium bicarbonate to (desalted) antibodies to make up a final concentration of 33 mM and adjust the pH of the solution to pH 8.
  2. Add to the reaction tube 15-fold molar excess of AcX and marinate on a roller for two hours at room temperature or overnight at 4 °C.
  3. Remove free AcX with a ZebaSpin desalting column according to the manufacturer’s instructions.
  4. Concentrate protein to approximately 2 mg / ml.
  5. Store at 4 °C. Any sodium azide added for storage should be removed before use as it can interfere with gel polymerisation.

REPLICA protocol

  1. Fix sample using 4 % paraformaldehyde.
  2. Wash sample three times in PBS for one hour each.
  3. Incubate the sample twice with PBS / 2 % Triton X-100 for one hour each.
  4. Incubate the sample with PBS / 2 % Triton X-100 / 20 % DMSO at 37 °C overnight.
  5. Incubate the sample with PBS / 0.5 % Tween-20 / 0.5 % Triton X-100 / 0.5 % deoxycholate / 0.5 % NP40 / 20 % DMSO at 37 °C overnight.
  6. Wash twice in PBS for one hour each.
  7. Block for one hour at room temperature using blocking buffer.
  8. Incubate the sample for at least 48 hours at 4 °C in blocking buffer with primary antibodies.
  9. Wash three times with PBS-Tween (0.1 %) for 30 minutes each.
  10. Incubate for at least 48 hours at 4 °C in blocking buffer with functionalised secondary antibodies.
  11. Wash three times with PBS-Tween (0.1 %) for 30 minutes to an hour each.
  12. Incubate the sample with preincubation monomer solution at 4 °C in an ice-cold water bath with very gentle shaking overnight.
  13. Incubate the sample with polymerisation monomer solution at 37 °C overnight.

Option A: tissues with low/no collagen (e.g. brain)

  1. Incubate with 8 units / ml proteinase K and 2 units / ml DNAse in enzyme digestion buffer at 37 °C with gentle shaking overnight.
  2. Incubate in cleaning buffer for at least one day at 37 °C with gentle shaking.
  3. Wash with 1 % w/v DGME for 30 minutes each, three times.
  4. Repeat steps 14–16 as appropriate until tissue is cleared.
  5. Wash with water as a refractive index matching liquid.

Option B: tissues with high collagen (e.g. kidney)

  1. Incubate with collagenase mix at 37 °C with gentle shaking for seven hours (timing can be adjusted to fit within working day).
  2. Incubate with 8 units / ml proteinase K and 2 units / ml DNAse in enzyme digestion buffer at 37 °C with gentle shaking overnight.
  3. Repeat steps 14–15 as appropriate until tissue no longer clears.
  4. Incubate in cleaning buffer for at least one day at 37 °C with gentle shaking.
  5. Repeat steps 14–17 as appropriate until tissue is cleared.
  6. Wash with water as a refractive index matching liquid.