Alexa fluor 594 donkey anti mouse

FFPE liver biopsy tissue slides were double stained for ubiquitin (Millipore, Temecula, CA) and the second protein as listed in Table 1. ubiquitin was detected using the second antibody donkey anti-mouse Alexa Fluor 594 (Jackson Labs, West Grove, PA), while the other protein was detected using the second antibody donkey anti-rabbit Alexa Fluor 488 or donkey anti-mouse Alexa Fluor 488 (Jackson Labs, West Grove, PA). All slides were stained with the nuclear stain DAPI (Molecular Probes, Eugene, OR). The fluorescence intensity of staining of the protein of interest was measured quantitatively using 40x objective magnification and a standard exposure time of 800 ms using a Nikon 400 fluorescent microscope with three filters (FITC-green, Texas-red, and tricolor), and the Nikon morphometric system. The results were displayed as a graph attached to the immunofluorescent photography using a screen snip. The staining was compared among MDB forming AH hepatocytes, neighboring non-MDB forming AH hepatocytes, and control normal hepatocytes.

Tissue slides were then prepared from the FFPE blocks. The slides were double stained for ubiquitin (Millipore, Temecula, CA) and either Mca1, Hsp104, Hsp40, Ydj1, Ssa1, VCP/p97, or p62. ubiquitin was detected using the red fluorescent(Texas Red) antibody, donkey anti-mouse Alexa Fluor 594 (Jackson Labs, West Grove, PA), while Mca1, Hsp104, Hsp40, Ydj1, Ssa1, VCP/p97, and p62 were detected using the green fluorescent(FITC) antibody, donkey anti-rabbit Alexa Fluor 488 or donkey anti-mouse Alexa Fluor 488 (Jackson Labs, West Grove, PA). The nuclei were stained with DAPI blue. The double stain was detected using a tricolor filter. All biopsies were stained at one time to allow accurate comparisons between groups.

Formalin fixed, paraffin embedded tissue slides were double stained for SYK (Abcam Inc., Cambridge MA) and Ubiquitin (Millipore, Temecula, CA). A second set of slides were stained for AKT1 (Invitrogen, Camarillo, CA) and Ubiquitin. SYK and AKT1 were detected using the second antibody donkey anti rabbit Alexa fluora 488 and anti-mouse Alexa fluora 594 (Jackson Labs, West Grove, PA) respectively. Ubiquitin was detected using the second antibody donkey anti mouse Alexa Fluor 594 (Jackson Labs. West Grove, PA). All slides were stained with the nuclear stain DAPI (Molecular Probes, Eugene, OR). The fluorescence intensity of stained protein of interest was measured quantitatively using a 40× objective and a standard exposure time of 800ms using a Nikon 400 fluorescent microscope with three filters (FITC-green, Texas Red, and Tri-Color), and the Nikon morphometric system (Liu et al., 2015 (link)).

Formalin fixed, paraffin embedded tissue slides were double stained for CXCR4 (Abcam Inc., Cambridge MA) and Ubiquitin (Millipore, Temecula CA). A second set of slides was double stained for CXCR7 (Millipore, Temecula CA) and Ubiquitin (Millipore, Temecula, CA. CXCR4 and CXCR7 were detected using the second antibody donkey anti rabbit Alexa Fluor 488 (Jackson Immuno Research Laboratories Inc. West Grove, PA). Ubiquitin was detected using the second antibody donkey anti mouse Alexa Fluor 594 (Jackson Labs. West Grove, PA). All slides were stained with the nuclear stain DAPI (Molecular Probes, Eugene, OR). The slides were examined with a Nikon 400 fluorescent microscope.

Upon completion of behavioral testing, all mice were deeply anaesthetized with Avertin (250 mg/kg; ip.) and transcardially perfused with ice cold saline (15 mL) followed by 4% paraformaldehyde (PFA; 15mL). Brains were extracted and post-fixed in PFA for 24 h. Tissue was sliced in 40 uM sections using a Vibratome and serial sections containing the entirety of the dSep were processed for immunohistochemistry to visualize the mCherry tag. Tissue was washed in 1xPBS, permeabilized in 0.5% Triton X-100 for 30 min, and blocked in 0.1% Triton X-100 and Normal Donkey Serum (NDS; 10%) for 1 h. Tissue was incubated for 24 h in the primary antibody solution that consisted of the blocking solution and mouse anti-RFP (1:500; Rockland Immunohistochemicals). The following day, tissue was washed in 1xPBS and incubated in a secondary solution that consisted of Alexa-Fluor 594 donkey anti-mouse (1:200; Jackson Immuno) in 0.1% Triton X-100 for 2 h. Tissue was washed in 1xPBS, mounted on Permafrost Plus slides, and coverslipped for Vectashield Hardset with DAPI. Slides were imaged on a Keyence microscope and viral expression was verified within the dSep through visualization of mCherry within the target region. A depiction of the general viral footprint and spread within the dSep is shown in Fig. 1A and a representative image of mCherry expression is shown in Fig. 1B .