Bond 3 automated ihc stainer

CTC and DTC cytospots were fixed in 10% buffered formalin (Leica Biosystems, Richmond VA), and stained with PHOX2B (1:100) (ab183741, Abcam, RRID : AB_2857845) and TH (1:3200) (66334-1-Ig, Proteintech, RRID : AB_2881714) with the BOND-III Automated IHC stainer (Leica Biosystems, USA), using manufacturers’ default automated staining protocol, as follows: pre-treatment unmasking with BOND epitope retrieval solution 2 (for PHOX2B) or 1 (for TH) (Cat. AR9640, AR9961, Leica Biosystems, USA), wash with absolute alcohol and Bond Wash Solution, staining with primary antibodies and detection using Bond™ Polymer Refine Detection (Cat. DS9800, Leica Biosystems, USA). After staining, slides were dehydrated in absolute alcohol, cleared with xylene and mounted in DEPEX medium.

Mice were sacrificed and eyes were fixed in Davidson’s fixative (deionized water, 10% acetic acid, 20% formalin, 35% ethanol) overnight, followed by dehydration in serial ethanols and embedding in paraffin blocks. Ten-μm thick microsections were cut along the horizontal meridian, progressively distributed on slides.
Paraffin-embedded mouse retinae were treated with a citrate buffer for antigen retrieval, incubated with primary antibodies, and developed with 3,3′-Diaminobenzidine (DAB) using a Bond-III Automated IHC Stainer from Leica Biosystems according to the manufacturer’s instructions. The primary antibodies used were as follows: monoclonal 1D4 (1:1,000, a gift from Robert Molday), monoclonal 4D2 (1:30,000, Millipore, Burlington, MA, USA), and monoclonal anti-GFP (1:2,000, Proteintech, Manchester, UK). Images were acquired in bright field using a Zeiss LSM 510 AxioCam microscope.

After euthanization, the Achilles tendon of each rat was dissected carefully from the attached calcaneal bone. The samples were fixed using 4% buffered formalin, embedded in paraffin, and stained with H and E and Alcian blue to quantify the severity of tendinopathy. Tissue changes were evaluated by histological criteria according to the Movin score. Images were obtained using a high-resolution Leica DC200 digital imaging system (Leica Microsystems, Wetzlar, Germany) mounted on an Olympus DMLB microscope. The Movin score was used to quantify the amount of tendon degeneration using H and E and Alcian blue staining (Supplementary Table S1) [36 (link),37 (link)]. Immunohistochemistry with SP antibody (ab14184, Abcam, Cambridge, MA, USA) was performed on 5-μm-thick formalin-fixed, paraffin-embedded blocks on a Bond-III Automated IHC Stainer (Leica Biosystems). The SP antibody was diluted (1:1000) and the BOND Polymer Refine Detection Kit was used according to the manufacturer’s instructions. ImageJ software (National Institute of Health, Bethesda, MD, USA) was used to quantify the amount of SP expression after immunohistochemical staining.

We designed a 9-plex immunohistochemistry panel [24 (link)], of which 6 markers were used in this analysis: Macrophages were identified using CD68 marker, and further phenotyping to M1 and M2 polarisation was performed with 4 markers [M1: HLADR and CD86; M2: CD163 and MRC1(CD206)]. Additionally, keratin (KRT) was used to detect tumour cells. We used the standardised nomenclature system for genes and gene products (www.genenames.org) to improve clarity and reduce ambiguity as recommended by the expert panel [25 ]. The multiplex immunohistochemistry staining was done with Bond-III automated IHC stainer (Leica Biosystems), Bond Refine Detection kit (DS9800, Leica Biosystems), and Dako AEC + High Sensitivity Substrate Chromogen (K3469, Agilent, Santa Clara, CA, USA) as previously described [24 (link)]. The procedure included staining one marker at a time, scanning the slide, heat-mediated antibody removal, and AEC removal with ethanol [24 (link)]. Candidate antibodies and suitable dilutions were optimised in a test tissue microarray consisting of normal colorectal mucosa, colorectal cancer tissue and tonsil tissue. These antibodies were then combined into a multiplex immunohistochemistry assay that was validated by confirming similar staining patterns of multiplex and conventional immunohistochemistry [24 (link)]. All slides were stained in one batch.