Multiplexed-ISH for Mycobacterium tuberculosis

Mouse lungs were fixed with formaldehyde, paraffin embedded, and stained by multiplexed-ISH using optimized protocols, as previously described (39 (link), 40 (link)). Briefly, mouse lungs were directly placed into 4% PFA prepared in 1× PBS upon excision, fixed for 48 h at 4°C, and paraffin embedded. Next, 2-μm sections were cut from formalin-fixed paraffin-embedded (FFPE) blocks and mounted onto Superfrost Plus microscope slides (Fisher Scientific). ISH for M. tuberculosis 23S and pre-rRNA was carried out by RNAscope (Advanced Cell Diagnostics), which was performed as described earlier (39 (link), 40 (link)). Sequences for the M. tuberculosis-rRNA ISH probes were previously described (40 (link)). Whole-slide digital images were acquired at ×40 magnification using an Axio Scan Z1 slide scanning fluorescence microscope (Zeiss).

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Robertson G.T., Ramey M.E., Massoudi L.M., Carter C.L., Zimmerman M., Kaya F., Graham B.G., Gruppo V., Hastings C., Woolhiser L.K., Scott D.W., Asay B.C., Eshun-Wilson F., Maidj E., Podell B.K., Vásquez J.J., Lyons M.A., Dartois V, & Lenaerts A.J. (2021). Comparative Analysis of Pharmacodynamics in the C3HeB/FeJ Mouse Tuberculosis Model for DprE1 Inhibitors TBA-7371, PBTZ169, and OPC-167832. Antimicrobial Agents and Chemotherapy, 65(11), e00583-21.

Publication 2021

Superfrost Plus microscope slides RNAscope Axio Scan Z1 slide scanning fluorescence microscope

Cell Diagnostics Embedded paraffin Fluorescence microscope Formaldehyde Formalin Lungs M tuberculosis Microscope Mouse Paraffin Pre rrna Rrna

Corresponding Organization : Colorado State University

Other organizations : Center for Discovery, Hackensack Meridian Health, University of California, San Francisco

Top 5 similar protocols

Variable analysis

independent variables

Formaldehyde fixation of mouse lungs
Paraffin embedding of mouse lungs
Multiplexed-ISH staining of mouse lung sections using optimized protocols

dependent variables

Expression of M. tuberculosis 23S and pre-rRNA in mouse lungs

control variables

Thickness of mouse lung sections (2-μm)
Use of Superfrost Plus microscope slides
Acquisition of whole-slide digital images at 40x magnification using an Axio Scan Z1 slide scanning fluorescence microscope

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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