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Materials and methods

This section details the materials and methods used to obtain the data presented in section 2.

FFPE tissues

Multiple tissues from three commonly used animals for preclinical studies (rat, dog, and cynomolgus monkey) were harvested using a standard protocol at the drug safety research and development laboratory of Pfizer Global Research and Development (Groton, USA) (Table 4). Tissues were cut into 3 mm thickness, then fixed in 10% neutral-buffered formalin (NBF) for 24-48 hours. Fixed tissues were dehydrated in a graded series of ethanol and xylene, followed by infiltration of melted paraffin at 56°C in an automated processor. Tissue microarrays (TMAs) were constructed, sectioned at a thickness of 5 μm and mounted on the SuperFrost® Plus slides (Fisherbrand Cat #12-550-15).

Animal Models: Rat, Dog, and Cynomolgus Monkey
Cardiovascular systemHeart
Endocrine glands/exocrine glandsLiver, Pancreas, Adrenal gland
GI tractEsophagus, Stomach, Duodenum, Jejunum, Colon
Hematopoietic systemThymus, Lymph node, Spleen, Tonsil
Nervous systemSpinal cord, Retina
Reproductive systemEpididymis, Prostate, Testis, Ovary
Respiratory systemLung, Bronchus
Skin/soft tissuesSkin, Skeletal muscle
Urinary tractKidney, Urinary bladder
TABLE 4. Tissue types from three animal models used in this study

Automated RNAscope® 2.5 LS assay

Ready-to-use reagents from RNAscope® 2.5 LS Reagent Kit-BROWN were loaded onto the Leica Biosystems’ BOND RX Research Advanced Staining System according to the user manual (Doc. No. 322100- USM). FFPE tissue sections were baked and deparaffinized on the instrument, followed by epitope retrieval (using Leica Epitope Retrieval Buffer 2 at 95°C or at 88°C for 15 min) and protease treatment (15 min at 40°C). Probe hybridization, signal amplification, colorimetric detection, and counterstaining were subsequently performed. A schematic of the RNAscope® 2.5 LS Assay workflow on Leica Biosystems’ BOND RX Research Advanced Staining System is presented in Figure 2.

RNAscope® probes

Control probes for low-, medium-, and high-expressing housekeeping genes (POLR2A, PPIB, and UBC, respectively) were tested for tissues from each species. Because the sequences of the human probes for housekeeping genes are over 95% homologous to the respective target mRNA sequences of cynomolgus monkey, human probes were used to test samples of cynomolgus monkey. The bacterial probe dapB was used as a negative control. Probes for the cell type biomarkers, proliferation markers, and apoptosis-related molecules used in this study were designed for each species. As summarized in Table 3, species-specific target probes were tested for all RNA targets except two genes, CD68 and KI67, for which human probes were used to detect cynomolgus monkey genes, due to 90-95% homology between the probe sequence and target mRNA sequence.

Image acquisition and data analysis

Images were acquired using a Leica Biosystems Aperio AT2 Digital Pathology Scanner, and RNA markers were analyzed based on the average RNA dot number per cell. RNA quantity was scored based on manual counting described as follows. Staining results were categorized into five grades according to the number of dots visualized under a bright-field microscope. 0: No staining or less than 1 dot to every 10 cells (40X magnification); 1+: 1-3 dots/cell (visible at 20-40X magnification); 2+: 4-10 dots/cell, very few dot clusters (visible at 20-40X magnification); 3+: >10 dots/cell, and less than 10% positive cells have dot clusters (visible at 20X magnification); and 4+: >10 dots/cell, and more than 10% positive cells have dot clusters (visible at 20X magnification). Figure 3 shows example of scoring for a variety of tissues, and Figure 6 (page 147) shows examples of gradual scoring.

Prostate, Score= 2+
Prostate, Score= 2+
Epididymis, Score= 3+
Epididymis, Score= 3+
Testis, Score= 2+
Testis, Score= 2+
Ovary, Score= 3+
Ovary, Score= 3+
Stomach, Score= 2-3+
Stomach, Score= 2-3+
Duodenum, Score= 3+
Duodenum, Score= 3+
Jejunum, Score= 3+
Jejunum, Score= 3+
Colon, Score= 2+
Colon, Score= 2+
Kidney, Score= 2+
Kidney, Score= 2+
Bladder, Score= 2+
Bladder, Score= 2+
Lymph node, Score= 2+
Lymph node, Score= 2+
Thymus, Score= 1-2+
Thymus, Score= 1-2+
Esophagus, Score= 2+
Esophagus, Score= 2+
Skin, Score= 2+
Skin, Score= 2+
Spinal cord, Score= 2+
Spinal cord, Score= 2+
Liver, Score=2+
Liver, Score=2+

 

FIGURE 3. Representative images for tissue qualification using the control probe Rn-Ppib in multiple rat tissues

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