Paraffin Tissue Preparation and Imaging

Paraffin sections were cut 5 μm thick, collected on Superfrost Plus Gold slides (Fisher Scientific), air-dried overnight, and baked for 1 hour at 60°C. Sections were processed for RNA in situ hybridization (ISH) with the RNAScope Detection Kit (Chromogenic) according to the manufacturer’s standard protocol (Advanced Cell Diagnostics, Hayward, CA) or as in a previous study [26 (link)]. Slide images were acquired using a Leica Aperio CS2 digital scanner and captured at 40x magnification with a resolution of 0.25 μm per pixel. For immunohistochemistry (IHC), sections were incubated with primary antibodies (1:500 dilution) at 4°C overnight as described in S1 Method. Following five washes, they were incubated with secondary antibodies (goat anti-mouse antibody Alexa Fluor 488; 1:500 dilution) for overnight. After five additional washes, they were coverslipped and imaged with a confocal laser scanning microscope (TCP SP2; Leica) or EVOS FL Auto Imaging System (ThermoFisher) at room temperature.

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Hu X., Steimel J.P., Kapka-Kitzman D.M., Davis-Vogel C., Richtman N.M., Mathis J.P., Nelson M.E., Lu A.L, & Wu G. (2019). Molecular characterization of the insecticidal activity of double-stranded RNA targeting the smooth septate junction of western corn rootworm (Diabrotica virgifera virgifera). PLoS ONE, 14(1), e0210491.

Publication 2019

Superfrost Plus Gold slides Aperio CS2 TCP SP2 EVOS FL Auto Imaging System

Alexa fluor 488 Anti antibody Antibodies Cell Chromogenic Confocal laser scanning microscope Diagnostics Dilution Goat Gold Immunohistochemistry In situ hybridization Mouse Paraffin Scanner

Corresponding Organization : Pioneer Hi-Bred

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Variable analysis

independent variables

Tissue section thickness (5 μm)
Tissue section preparation (collected on Superfrost Plus Gold slides, air-dried overnight, baked for 1 hour at 60°C)
RNA in situ hybridization (ISH) protocol (RNAScope Detection Kit (Chromogenic) with or without a previous study [26])
Primary antibody dilution (1:500)

dependent variables

Gene expression (measured using RNA in situ hybridization)
Protein expression (measured using immunohistochemistry)

control variables

Tissue section thickness (5 μm)
Tissue section preparation (collected on Superfrost Plus Gold slides, air-dried overnight, baked for 1 hour at 60°C)
Imaging parameters (slide images acquired using Leica Aperio CS2 digital scanner at 40x magnification with 0.25 μm per pixel resolution, confocal laser scanning microscope (TCP SP2; Leica) or EVOS FL Auto Imaging System (ThermoFisher) at room temperature)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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