Mayer s hematoxylin

Tissue sections were adhered to the ST arrays at 37°C for 1 min, in situ fixated in 4% PFA (Sigma Aldrich, USA) at RT and washed in 50mL 1x PBS (Gibco). In most cases, 4 tissue sections were fitted onto one ST active area. Tissues were then dried for 1min in isopropanol, followed by hematoxylin and eosin (H&E) staining. Briefly, tissue sections were exposed to 100% Mayer’s hematoxylin (DAKO, Agilent) for 6 minutes followed by washing for 2 min in deionized water at RT. To adjust the pH, slides were briefly dipped in DAKO’s Bluing buffer (Agilent) and then counterstained in 5% eosin diluted in Tris-AA (pH 7.2) for 1 min. Slides were again washed in deionized water and dried prior to mounting them with 85% glycerol prior to imaging. All samples were imaged on an Axio Imager Z2 microscope equipped with a 20x/0.8 Plan-APOCHROMAT (Carl Zeiss Microscopy, Germany) and the resulting images stitched with Vslide (v1.0.0, MetaSystems GmbH).

Sectioned slides were incubated at 37 °C for 1 min and fixed in methanol for 10 min at −20 °C. For staining, the sections were incubated in isopropanol (MilliporeSigma, Burlington, MA, USA) for 6 min, Mayer’s hematoxylin (Agilent, Santa Clara, CA, USA) for 7 min, Bluing Buffer (Dako) for 1 min, and eosin (Sigma-Aldrich, St. Louis, MO, USA) diluted 1:5 in Tris-base (0.45 M Tris, 0.5 M acetic acid, pH 6.0) for 1 min. The slides were washed with deionized water after each of the staining steps. After air-drying, the slides were mounted with 85% glycerol and coverslips. Hematoxylin and eosin-stained images were recorded at 40× magnification using a digital slice scanner (Hamamatsu, San Jose, CA, USA). The coverslip was removed after imaging by immersing the slides in RNase-and DNase-free water.

Immunohistochemistry (IHC) analyses were performed as in [Iachettini 2023]. After sectioning and processing, the tissue sections were immunostained for 1 hours at RT with anti-TERF2/TRF2 rabbit polyclonal (1:500), anti-LC3B rabbit monoclonal (AbCam, Cambridge, United Kingdom, EPR21234, 1:100), anti-p62 SQSTM1 mouse monoclonal (D-3) (Santa Cruz Biotechnology, Dallas, Texas, USA, sc-28359, 1:300), anti-Ki67 mouse monoclonal (Agilent Dako, Santa Clara, CA, USA, MIB-1, 1:100), and anti-Cleaved Caspase-3 rabbit polyclonal (Cell Signaling, #9661, 1:100) antibodies and then were covered for 30 minutes at RT with Dako EnVision™ FLEX /HRP (EnVision™ FLEX; Agilent, Santa Clara, CA, USA, K8023). The signal was developed by using DAB detection kit (Agilent Dako, GV825), then sections were counterstained with Mayer’s Hematoxylin (Agilent Dako, S3309). Finally, slides were washed, dehydrated and mounted with Eukitt (Sigma-Aldrich, 03989). Immunostaining results were recorded as percentage of positive cells or immunoreactive score (IRS, staining intensity per percentage of positive cells).

Formalin-fixed and paraffin-embedded
(FFPE) tissues were cut into 3–4 μm sections and put
on FLEX IHC microscope slides (K8020, DAKO). Slides were heated at
60 °C for 60 min and deparaffinized in xylene (2 × 10 min).
Rehydration was performed in decreasing concentrations of ethanol
(100% ethanol: 1 × 5 min, 95% ethanol: 1 × 5 min) followed
by rinsing in distilled water. The immunohistochemical (IHC) staining
for KI67 was performed using an Autostainer Plus (DAKO) instrument.
Antigen retrieval was performed on a PT-LINK (Agilent) instrument
using the EnVision FLEX target retrieval solution (pH 9, dilution:
1:10) at 98 °C for 20 min. Slides were stained by incubating
the primary antibody (Ki67:clone MIB-1, M7240, Agilent Technologies)
at the following dilution: 1:200 (temperature: RT, time: 30 min).
The antibody–antigen complex was visualized using the EnVision
FLEX DAB detection kit (K801021-2, Agilent Technologies) and counterstained
with Mayer’s hematoxylin (S3309, Agilent Technologies). Stained
slides were dehydrated in increasing concentrations of ethanol (95%
ethanol: 1 × 3 min, 100% ethanol: 1 × 3 min), followed by
xylene (2 × 5 min). Cover glasses were mounted using a Coverslipper
DAKO (Agilent Technologies), and slides were left to dry prior to
staining evaluation.

Slides of 4 μm paraffin-embedded liver tissue sections were deparaffinized and rehydrated. After washing, the slides were boiled with 10 mM sodium citrate (pH 6.0) (ICN biomedicals, Costa Mesa, CA, USA) for antigen retrieval and then treated with 0.5% Triton X-100 (Yakuri pure chemicals, Kyoto, Japan) in Tris-buffered saline (TBS) for 5 min to permeabilize for antibodies.
Antibodies against transforming growth factor-beta (TGF-β) 1 (1:50 dilution, ab92486, Abcam), alpha-smooth muscle actin (α-SMA) (1:100 dilution, ab7817, Abcam), and F4/80 (1:100 dilution, ab6640, Abcam) were diluted with TBS containing 1% BSA and applied overnight at 4 °C. Next, the slides were incubated with secondary antibodies of donkey anti-rat IgG Alexa Fluor 488 (1:500 dilution, A-21208, Thermofisher Scientific, Waltham, MA, USA), goat anti-mouse IgG Alexa Fluor 488 (1:500 dilution, A-11001, Thermofisher Scientific), and donkey anti-rabbit IgG Alexa Fluor 555 (1:500 dilution, A-31572, Thermofisher Scientific) for 1 h. Finally, the slides were DAPI stained and mounted.
For H&E staining, the slides were deparaffinized, rehydrated, and treated with Mayer’s hematoxylin (Agilent Technologies) for 20 s and washed. Next, eosin solution (Sigma-Aldrich) was added to the slides for 30 s and washed. Finally, the slides were mounted.