Mice
Breeding pairs of Hps1 gene mutant (Hps1ep/ep) mice on the C57BL/6 J background were kindly provided by Dr. Susan H. Guttentag, University of Pennsylvania. C57BL/6 J mice were used as wild type (WT) controls. Procedures on Hps1ep/ep mice were conducted on 8–12 weeks old male mice. Mice were bred in a pathogen-free barrier facility in accordance with NIH guidelines and approved by the NHGRI Animal Care and Use Committee (ACUC), under the protocol G-14-3 “Mouse models for disorders of lysosomes and lysosomal-related organelles”.
Administration of bleomycin
Oropharyngeal administration of bleomycin, which allows direct aspiration of bleomycin, was performed by an expert scientist as previously described [16] with slight modifications to the dose based on the increased sensitivity of Hps1ep/ep to bleomycin. Mice were placed on an adjustable stand and an external light source was directed over the trachea to visualize the vocal cords. Bleomycin (Hospira Inc.) was delivered at doses of 0.1 (n = 14), 0.2 (n = 14), 0.3 (n = 14), 0.5 (n = 13) or 1 U/Kg (n = 13). For control, mice were given saline (n = 6). Mice were followed for 14 days and their body weight was recorded; mice that lost 25% of baseline body weight were euthanized.
Systemic administration of bleomycin by subcutaneous osmotic minipump was performed as previously described [17]. Bleomycin was delivered continuously at 0 (n = 18), 15 (n = 5), 30 (n = 5), 45 (n = 13) or 60 (N = 26) U/Kg in 0.9% saline in the suprascapular subcutaneous space of Hps1ep/ep mice via a surgically implanted 1007D Azlet osmotic minipumps on days 0 to 7. C57BL/6 J wild type were challenged with 0 (n = 6) or 100 U/Kg (n = 26). On day 7, minipumps were removed. Mice were euthanized at 0, 14, 21, 28, or 42 days post minipump implantation.
Histopathology
Mice were euthanized using 100–150 μL of intraperitoneal 1.25% tribromoethanol unless specified otherwise. After euthanasia, the chest cavity was opened and all lobes of the right lung were inflated with 500 μL of 10% formalin intratracheally, removed, and immersed in formalin. Skin tissue taken from regions distant from the suprascapular area, kidney, and lung tissues were embedded in paraffin, sectioned at 5 µm, and stained with haematoxylin and eosin (H & E), and Masson’s trichrome. Bright field photomicrographs of stained tissues were imaged randomly without area selection using a Zeiss AxioObserver Z1 widefield microscope equipped with a plan-apochromat (N.A. 0.45) objective lens, a motorized scanning stage, and an Axiocam MRc5 color CCD camera (Zeiss).
Hydroxyproline quantification
Prior to right lung inflation, the left main bronchus was ligated, and the left lung was excised, snap frozen in liquid nitrogen and stored at – 80 °C. Hydroxyproline quantification was performed via LC–MS/MS as previously described [16]. Assays were done in technical duplicates.
Fluorescent in situ hybridization (FISH)
RNAscope® Multiplex Fluorescent Kit v2 (Advanced Cell Diagnostics (ACD), Newark, CA, USA) was used to perform RNA-probe based fluorescent in situ hybridization (FISH) on 5 µm thick formalin fixed paraffin embedded (FFPE) mouse lung tissue. Lung tissue sections were baked at 60 ºC for 60 min, deparaffinized in two 5 min washes of Hemo-De® (VWR, Randor, PA, USA) followed by two 5 min washes of 100% ethanol, and then air-dried at room temperature for 10 min. Endogenous peroxidases were quenched with a 10 min incubation with hydrogen peroxide and protease (RNAscope®, ACD, Cat. 322330) at room temperature, and sections were washed twice with water. Antigen retrieval was performed by steaming lung tissue at 99–100 ºC in water for 10 s, then 1X RNAscope® Target Retrieval (ACD, Cat. 322000) for 15 min. Slides were then moved immediately to water for 15 s, to 100% ethanol for 3 min and then air dried. Hydrophobic barrier was drawn (PAP pen, Abcam, Cat. ab2601), then 5 drops of Protease Plus (ACD, Cat. 322331) were added to digest the lung tissue at 40º for 30 min; slides were then washed twice with water.
RNAscope® ISH probes from ACD were used at their recommended dilutions and incubated on lung tissue at 40 ºC for 120 min. The probes used from ACD were TGF-β in channel 1 (ACD, 322331), Il1-β in channel 2 (316891-C2), and Polr2a in channel 3 for control (312471-C3). The samples were washed twice in 1X RNAscope® Wash Buffer (31009) and stored in 5X SSC buffer (Quality Biological, 351003101) overnight. Slides were washed twice with washing buffer and treated with Amp 1–3 (323100) according to the manufacturer’s instructions. The fluorophores (Perkin Elmer, Waltham, MA, USA) used to visualize the probes were fluorescein (NEL741E001KT), cyanine 3 (NEL744001KT), and cyanine 5 (NEL745E001KT), which were diluted 1:750 in a TSA buffer (ACD, 322810). Slides were treated with HRP-C1-3 to develop a signal, then fluorophore (C1-fluorescein, C2-Cyanine 3, C3-Cyanine 5) and HRP blocker (ACD, 323100) as specified in the manufacturer’s protocol. Slides were treated with DAPI for 30 s and mounted with VECTASHIELD® Antifade Mounting Medium (Vector laboratories, Burlingame, CA, USA; Cat. H-1000-10) prior to applying a coverslip.
After 24 h, random fields in peripheral and central lung regions were imaged using a Zeiss 510 META confocal laser-scanning microscope (Carl Zeiss, Thornwood, NY, USA). All in situ experiments were performed for data collection in technical duplicates. Several optimization steps were condeucted before final data collection.
Data analysis
The RNAScope data were analyzed using ZEN Blue software and the Image Analysis Wizard. The analysis parameters were set using a small subset of images and later validated on a larger set. The approximate accuracy was determined by visual inspection of the mask overlayed on the microscope image. The final analysis routine described in Additional file 3: Table S1 was saved into an Image Analysis Settings file. This file was loaded as a Settings parameter of the “Analyze Batch to File” processing option in ZEN. The analysis was performed in the Single function mode.