Benchmark special stain system

A single 4 µm paraffin section from each sample (i.e., containing both wedge-shaped biopsy specimens) was stained with Haematoxylin (Harris Haematoxylin, Shandon, UK) and Eosin (Eosin Y, Shandon, UK) (H&E) using an autostainer (Leica ST5020, Milton Keynes, UK).
One additional section from each sample was stained with picro-Sirius red (SR) stain (Sigma-Aldrich Direct Red 80, Dorset, UK) with the following protocol: (1) deparaffinize sections in 2 changes of xylene; (2) rehydrate through 2 changes of alcohol (70% alcohol and distilled water); (3) apply SR solution to cover entire sections and incubate in humidified chamber for 60 min; (4) after 60 min, rinse slides quickly in 2 changes of Acetic Acid Solution; and (5) rinse slides in absolute alcohol and then dehydrate through 2 changes of alcohol, clear in 2 changes of xylene, and mount with pertex mounting medium.
A further section from each sample was stained for reticulin. The staining procedure was carried out using the Reticulum II stainer kit (Roche catalogue number 860-024) and a Ventana BenchMark Special Stain system.

Haematoxylin & eosin (H&E) staining of the excised histotripsy sites was used to identify important structural information and any change following sonication to the liver parenchyma.
Picrosirius red stain was used to demonstrate structural and architectural damage to collagen following histotripsy by staining for collagen type I & III. A single 4 µm paraffin section from each sample was stained with haematoxylin (Harris Haematoxylin, Shandon, UK) and eosin (Eosin Y, Shandon, UK) (H&E) using an auto-stainer (Leica, ST5020, Milton Keynes, UK).
One additional section from each sample was stained with Picrosirius red (SR) stain (Sigma-Aldrich, Direct Red 80, Dorset, UK) with the following protocol: (1) deparaffinise sections in two changes of xylene. (2) Rehydrate through two changes of alcohol, 70% alcohol and distilled water. (3) Apply SR solution to cover entire sections and incubate in humidified chamber for 60 min. (4) After 60 min, rinse slides quickly in two changes of acetic acid solution. (5) Rinse slides in absolute alcohol and then dehydrate through two changes of alcohol, clear in two changes of xylene and mount with pertex mounting medium.
A further section from each sample was stained for reticulin. The staining procedure was carried out using the Reticulum II stainer kit (Roche catalogue number 860–024) and a Ventana BenchMark Special Stain system.