Knockdown and subsequent complementation of tmRNA was confirmed by RT-qPCR following induction or not. Samples were infected and then induced or not. RNA and gDNA were extracted, processed, and analyzed as describe above, where transcript abundances were compared with the levels of gDNA. Additionally, strains were monitored for morphological differences by IFA. Samples were fixed with MeOH to eliminate GFP fluorescence and stained with goat anti-MOMP (Meridian), rabbit anti-Sa_dCAS9 (Abcam), and mouse anti-6×His (GenScript) primary antibodies. Samples were then stained with the appropriate secondary antibodies (Invitrogen) and DAPI (Sigma), mounted using ProLong glass antifade mounting media (Invitrogen), and imaged as described previously. The image shown is representative of three biological replicates.
Prolong glass antifade mounting media
Manufactured by Thermo Fisher Scientific
ProLong glass antifade mounting media is a ready-to-use solution designed for the preservation and stabilization of fluorescent signals in microscopy samples. It is formulated to reduce photobleaching and maintain the integrity of fluorescent dyes.
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5 protocols using prolong glass antifade mounting media
1
Confirming tmRNA Knockdown via RT-qPCR
2
Immunohistochemistry of Coronal Brain Sections
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Coronal brain sections were blocked with 2% BSA, 10% normal goat serum, 0.1% Triton, TBS (1h, RT), and incubated with primary antibodies diluted in blocking solution overnight at 4°C. After washing, antigen-bound primary antibodies were detected with the corresponding labelled Alexa 488, Alexa 555 or Alexa 647 secondary antibodies (1/300 – 1/500, Invitrogen) that were incubated for 1h at RT. Sections were then mounted either with ProLong glass antifade mounting media (Invitrogen) for super-resolution microscopy or with Vectashield Hardset (Vector) for confocal imaging.
3
Immunofluorescence Staining Protocol
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Sections were permeabilized and blocked with 10% normal donkey serum and 0.2% Triton X-100 plus 0.02% sodium azide in PBS (PBSTx azide) for 1 hour before the primary antibodies were applied (Table 1 ) for ~48 h. Prior to application of the secondary antibodies (Table 1 ), the slides were washed three times (5 min each) with PBS. Secondary antibodies were applied overnight at room temperature and subsequently washed 3 times for 5 min each in PBS. The refractive index was matched to glass using Cubic-R+(M) (TCI, Cat#T3741) in one 20-min application. Slides were coverslipped using 24x50mm coverslips and the Cubic mounting media (TCI, Cat# M3294) before being sealed with nail varnish. Slides with 50 micron-thick sections were not cleared with Cubic reagents, and instead coverslipped using ProLong Glass anti-fade mounting media (Invitrogen, P36984).
4
Immunofluorescence Labeling of Brain Sections
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Coronal brain sections were blocked with 2% bovine serum albumin (BSA), 10% normal goat serum, 0.1% Triton, TBS (1 h, room temperature [RT]), and incubated with primary antibodies diluted in blocking solution overnight at 4°C. After washing, antigen‐bound primary antibodies were detected with the corresponding labelled Alexa 488, Alexa 555, or Alexa 647 secondary antibodies (1/300 – 1/500, Invitrogen) that were incubated for 1 h at RT. Sections were then mounted either with ProLong glass antifade mounting media (Invitrogen) for super‐resolution microscopy or with Vectashield Hardset (Vector) for confocal imaging.
5
Histological Analysis of Transduced Tissues
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Mice were euthanized and perfused with PBS and freshly prepared 4% PFA (Sigma-Aldrich, P6148) before organ collection. Liver and heart were collected and placed into 4% PFA in PBS for 72 h before embedding. Brain was equilibrated with 15% sucrose + 4% PFA + PBS followed by 30% sucrose + 4% PFA + PBS solution before embedding. Samples were sent to Stanford Animal Histology Services for embedding in OCT media, sectioning at 5–10 µm and mounting on slides. Tissue was stained with Hoechst 33342, Trihydrochloride, Trihydrate (Fisher Scientific, H3570) at 1 µg ml−1 for 30 min. Coverslips were mounted using Prolong Glass Antifade Mounting Media (Invitrogen, P36980 ).
Slides were imaged on a Leica DM4 Upright Microscope (Canary Center at Stanford). AAV transduction was measured by EGFP fluorescence and AAVR-mCh was measured by mCherry expression, and Hoechst staining for the nucleus.
Slides were imaged on a Leica DM4 Upright Microscope (Canary Center at Stanford). AAV transduction was measured by EGFP fluorescence and AAVR-mCh was measured by mCherry expression, and Hoechst staining for the nucleus.
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