Click-iT HPG Alexa Fluor 594 protein synthesis assay kit (Life Technologies, C10429) was used to detect newly synthesized proteins in iKPC cells. Images were captured using a Leica inverted microscope system (DMI8) and the signal intensity in the fluorescent channel was determined by ImageJ. The corrected total cell fluorescence [=integrated density − (area of selected cell × mean fluorescence of background readings)] was determined from 10 fields that were randomly selected from different regions across the entirety of each sample.
Dmi8 inverted microscope system
Manufactured by Leica
The Leica DMi8 is an inverted microscope system designed for advanced imaging applications. It features a modular and flexible design, allowing for customization to meet the specific needs of researchers and scientists. The core function of the DMi8 is to provide high-quality, high-resolution imaging capabilities for a wide range of samples and applications.
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Lab products found in correlation
Cy3 conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
Prolong diamond antifade with dapi, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
1832 rbpms, by PhosphoSolutions (2 mentions)
Click it hpg alexa fluor 594 protein synthesis assay kit, by Thermo Fisher Scientific (1 mentions)
D1818, by Thermo Fisher Scientific (1 mentions)
Vectashield antifade mounting medium with dapi, by Vector Laboratories (1 mentions)
Sa00013 4, by Proteintech (1 mentions)
2 4 amidinophenyl 6 indolecarbamidine dihydrochloride dapi, by Beyotime (1 mentions)
9 protocols using dmi8 inverted microscope system
1
Quantifying Newly Synthesized Proteins
2
Endocytic Tracer Dextran Uptake
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Cells were washed once in warmed PBS before incubation in serum-free RPMI medium with 1 mg/mL 70,000-MW TMR-dextran (Invitrogen D1818) for 30 min at 37°C. After incubation, cells were washed five times in cold PBS on ice and fixed in 4% polyformaldehyde promptly for 15 min. Cells were washed for another three times before mounting in VectaShield antifade mounting medium with DAPI (Vector Laboratories H-1200-10). Images were captured using a Leica inverted microscope system (DMI8) and analyzed using the “analyze particles” feature in ImageJ. The total particle area per cell was determined from three to six fields that were randomly selected from different regions across the entirety of each sample.
3
Immunostaining of Brain Slices for NRF2
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The brain slices were rinsed three times with PBS for 5 min each and blocked in 5% BSA for 1 h. The primary antibody (rabbit anti-NRF2 antibody, 1:500,16396–1-AP, Proteintech) was incubated with the slices overnight at 4 °C. The next day, the slices were rewarmed in the primary antibody for 1 h at 24±1 °C and then washed three times for 5 min each time with 0.3% PBST. The secondary antibody (goat anti-rabbit IgG, 1:500, SA00013–4, Proteintech) was incubated at room temperature for 1 h in a wet box. Then, slices were washed in 0.3% PBST three times for 5 min each time and stained with 2–4-Amidinophenyl-6-indolecarbamidine dihydrochloride (DAPI, P0131, Beyotime) for 5 min. Fluorescence was captured on a LEICA DMI8 inverted microscope system.
4
Adipocyte Lipid Droplet Imaging
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At the end of adipogenesis, media was aspirated, and adipocytes were fixed with 4% paraformaldehyde for 45 minutes. Adipocytes were washed 3X with 1X TBS for 5-minutes. Adipocytes were permeabilized using 0.3%TritonX-100 in 1X TBS for 30 minutes. Adipocytes were washed 3X with 1X TBS for five minutes/wash. Adipocytes were incubated with HSC LipidTox-deep red or green (1/1000 in 1X TBS) for 45 minutes rocking in the dark. Adipocytes were washed twice with 1X TBS for five minutes/wash. Adipocytes were then stained with Hoechst (1 μg/ml in 1X TBS) for 10 minutes. Adipocytes were washed twice with 1X TBS for five minutes/wash. Fluorescent images were collected on a Leica DMi8 inverted microscope system.
5
Adipocyte Visualization and Quantification
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At the end of differentiation, media was aspirated, and adipocytes were fixed with 4% paraformaldehyde for 45 min. Adipocytes were washed thrice with 1X TBS for 5 min. Adipocytes were permeabilized using 0.3%TritonX-100 in 1X TBS for 30 min. Adipocytes were washed thrice with 1X TBS for 5 min/wash. Adipocytes were incubated with HSC LipidTox-deep red (1/1000 in 1X TBS) for 45 min rocking in the dark. Adipocytes were washed twice with 1X TBS for 5 min/wash. Adipocytes were then stained with Hoechst (1 µg/ml in 1X TBS) for 10 min. Adipocytes were washed twice with 1X TBS for 5 min/wash. Fluorescent images were collected on a Leica DMi8 inverted microscope system.
6
Histological Slide Rehydration and Staining
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Slides were rehydrated using the following protocol: xylene (3 minutes 3X), 100% reagent alcohol (1 minutes 2X); 95% reagent alcohol (1 min 2X); water (1 minutes). Slides were stained in hematoxylin and eosin (H&E) staining for 2:30 minutes and 10 repeated submerges, respectively. Slides were dehydrated in the reverse order of rehydration steps. Coverslips were mounted with Cytoseal 60 mounting media. Brightfield images were acquired using a Leica DMi8 inverted microscope system.
7
RGC Immunocytochemistry Protocol
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RGCs were cultured for 7 days in vitro (DIV) on 12mm coverslips before being fixed in 4% PFA for 15 mins at room temperature (RT) and then permeabilized in 0.1% Triton X-100 for 5 mins. Cells were then blocked for 1 hour at RT with 5% BSA and 5% normal donkey serum. Primary antibodies incubation with guinea pig anti-RBPMS (pan RGC marker) (1:250 dilution, #1832-RBPMS; PhosphoSolutions, Aurora, CO) and mouse anti-TUJ1 (neuron marker) (1:300 dilution, #MMS-435P; San Diego, CA) were performed overnight in a moist chamber at 4 °C. Cells were incubated with Cy3 conjugated secondary antibody (1:1000 dilution, #706-165-148; Jackson ImmunoResearch Laboratories, West Grove, PA) and Alexa Fluor 488 conjugated secondary antibody (1:1000 dilution, #A-21202; Thermo Fisher Scientific, Waltham, MA) for 1 hour at RT. Stained coverslips were sealed on to glass slides with Prolong Diamond antifade with DAPI (#P36971; Thermo Fisher Scientific, Waltham, MA). Six images per coverslips, in a fixed 3x2 grid, were acquired on a Leica DMi8 inverted microscope system (Buffalo Grove, IL). To determine RGC culture purity, RBPMS staining counts were compared to the overlap of co-labeled and only labeled DAPI positive stained cells. Immunocytochemistry of RGC RBPMS staining was performed from three isolations with a total cell count of n = 11,687.
8
H&E Staining and Brightfield Imaging
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Slides were rehydrated using the following protocol: xylene (3 min 3X), 100% reagent alcohol (1 min 2X); 95% reagent alcohol (1 min 2X); water (1 min). Slides were stained in hematoxylin and eosin (H&E) staining for 2 min 30 sec and 10 repeated submerges, respectively. Slides were dehydrated in the reverse order of rehydration steps. Coverslips were mounted with Cytoseal 60 mounting media. Brightfield images were acquired using a Leica DMi8 inverted microscope system.
9
Immunocytochemistry of Retinal Ganglion Cells
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RGCs were cultured for 7 days in vitro (DIV) on 12mm coverslips before being fixed in 4% PFA for 15 mins at room temperature (RT) and then permeabilized in 0.1% Triton X-100 for 5 mins. Cells were then blocked for 1 hour at RT with 5% BSA and 5% normal donkey serum. Primary antibodies incubation with guinea pig anti-RBPMS (pan RGC marker) (1:250 dilution, #1832-RBPMS; PhosphoSolutions, Aurora, CO) and mouse anti-TUJ1 (neuron marker) (1:300 dilution, #MMS-435P; San Diego, CA) were performed overnight in a moist chamber at 4 °C. Cells were incubated with Cy3 conjugated secondary antibody (1:1000 dilution, #706-165-148; Jackson ImmunoResearch Laboratories, West Grove, PA) and Alexa Fluor 488 conjugated secondary antibody (1:1000 dilution, #A-21202; Thermo Fisher Scientific, Waltham, MA)
for 1 hour at RT. Stained coverslips were sealed on to glass slides with Prolong Diamond antifade with DAPI (#P36971; Thermo Fisher Scientific, Waltham, MA). Six images per coverslips, in a fixed 3x2 grid, were acquired on a Leica DMi8 inverted microscope system (Buffalo Grove, IL). To determine RGC culture purity, RBPMS staining counts were compared to the overlap of co-labeled and only labeled DAPI positive stained cells. Immunocytochemistry of RGC RBPMS staining was performed from three isolations with a total cell count of n=11,687.
for 1 hour at RT. Stained coverslips were sealed on to glass slides with Prolong Diamond antifade with DAPI (#P36971; Thermo Fisher Scientific, Waltham, MA). Six images per coverslips, in a fixed 3x2 grid, were acquired on a Leica DMi8 inverted microscope system (Buffalo Grove, IL). To determine RGC culture purity, RBPMS staining counts were compared to the overlap of co-labeled and only labeled DAPI positive stained cells. Immunocytochemistry of RGC RBPMS staining was performed from three isolations with a total cell count of n=11,687.
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