After the exposure, the TT1 and AT2 cells were rinsed twice with PBS, fixed with 4% paraformaldehyde in PBS, and permeabilised with 0.1% triton X-100 with 0.5% FCS in PBS. Samples were then rinsed again with PBS three times for 5 min and incubated with 1% BSA in PBS for 40 min before incubating with primary antibody of EEA1, LAMP1, actin or ZO-1 (1:100; Rabbit polyconal IgG against EEA1, LAMP1 or ZO-1 from Invitrogen, UK) in 1% BSA overnight at 4°C in dark. The next day cells were rinsed (x3) and incubated with secondary antibody (1:200; Alex Fluor®594 - goat anti rabbit IgG from Invitrogen, UK) in 1% BSA in PBS for 1h at room temperature. Cells were then counter stained with DAPI and rinsed with PBS. Finally, membrane were cut and mounted onto the microscope slides using Prolong® Gold (Invitrogen, UK), kept at 4–8°C before imaging using the laser scanning confocal microscopy (Ziess LSM-510 inverted confocal microscopy, Germany). Three separate experiments were carried out with 50 cells surveyed (in total 150 cells were surveyed).
Alex fluor 594 goat antirabbit igg
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
AlexaFluor 594 goat anti-rabbit IgG is a secondary antibody conjugated with the AlexaFluor 594 fluorescent dye. It is used to detect and visualize rabbit primary antibodies in various immunological techniques, such as immunofluorescence microscopy, flow cytometry, and Western blotting.
Automatically generated - may contain errors
3 protocols using alex fluor 594 goat antirabbit igg
1
Immunofluorescence Analysis of Endocytic Markers
2
Oxidative Stress Induces ROCK1 and p53 Expression
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The B3 cells were cultured in 24-well plates and treated with 200 µM H 2 O 2 in media for 24h. These cells were xed with 4% paraformaldehyde for 10 minutes, permeabilized in 0.3% Triton X-100 for 10 minutes, blocked with 3% bovine serum albumin (BSA) for 30 minutes at 37℃. The primary antibodies of ROCK1(1:200, Abcam) and p53 (1:200, Cell Signaling Technology) were rst incubated at 37℃ for 1h and then at 4℃ overnight. Alex Fluor 594 goat antirabbit IgG (Invitrogen, A11037) and Alex Fluor 488 goat anti-mouse IgG (Invitrogen, A11029) were as the secondary antibodyy incubated for 1h at 37℃. Images were selected using a uorescence microscope.
RNA extraction, reverse transcription, and quantitative RT-PCR Total cellular RNA was extracted from B3 cells using the Promega Total RNA Isolation System according to the manufacturer's instructions. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using the Access RT-PCR System (Promega). The ROCK1 gene primer pairs for quantitative PCR assay were as follows: sense, 5′-TGTGACTGGTGGTCGGTT-3′ and antisense, 5-′GGTTTTTTGCTTCTTTT′G-3. Primer pairs for β-actin were as follows: sense, 5′-TCGTGCGTGACATTAAGGAG-3′, and antisense, 5′-ATGCCAGGGTACATGGTGGT′-3.
RNA extraction, reverse transcription, and quantitative RT-PCR Total cellular RNA was extracted from B3 cells using the Promega Total RNA Isolation System according to the manufacturer's instructions. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using the Access RT-PCR System (Promega). The ROCK1 gene primer pairs for quantitative PCR assay were as follows: sense, 5′-TGTGACTGGTGGTCGGTT-3′ and antisense, 5-′GGTTTTTTGCTTCTTTT′G-3. Primer pairs for β-actin were as follows: sense, 5′-TCGTGCGTGACATTAAGGAG-3′, and antisense, 5′-ATGCCAGGGTACATGGTGGT′-3.
3
Oxidative Stress Induces ROCK1 and p53 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The B3 cells were cultured in 24-well plates and treated with 200 µM H 2 O 2 in media for 24h. These cells were xed with 4% paraformaldehyde for 10 minutes, permeabilized in 0.3% Triton X-100 for 10 minutes, blocked with 3% bovine serum albumin (BSA) for 30 minutes at 37℃. The primary antibodies of ROCK1(1:200, Abcam) and p53 (1:200, Cell Signaling Technology) were rst incubated at 37℃ for 1h and then at 4℃ overnight. Alex Fluor 594 goat antirabbit IgG (Invitrogen, A11037) and Alex Fluor 488 goat anti-mouse IgG (Invitrogen, A11029) were as the secondary antibodyy incubated for 1h at 37℃. Images were selected using a uorescence microscope.
RNA extraction, reverse transcription, and quantitative RT-PCR Total cellular RNA was extracted from B3 cells using the Promega Total RNA Isolation System according to the manufacturer's instructions. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using the Access RT-PCR System (Promega). The ROCK1 gene primer pairs for quantitative PCR assay were as follows: sense, 5′-TGTGACTGGTGGTCGGTT-3′ and antisense, 5-′GGTTTTTTGCTTCTTTT′G-3. Primer pairs for β-actin were as follows: sense, 5′-TCGTGCGTGACATTAAGGAG-3′, and antisense, 5′-ATGCCAGGGTACATGGTGGT′-3.
RNA extraction, reverse transcription, and quantitative RT-PCR Total cellular RNA was extracted from B3 cells using the Promega Total RNA Isolation System according to the manufacturer's instructions. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using the Access RT-PCR System (Promega). The ROCK1 gene primer pairs for quantitative PCR assay were as follows: sense, 5′-TGTGACTGGTGGTCGGTT-3′ and antisense, 5-′GGTTTTTTGCTTCTTTT′G-3. Primer pairs for β-actin were as follows: sense, 5′-TCGTGCGTGACATTAAGGAG-3′, and antisense, 5′-ATGCCAGGGTACATGGTGGT′-3.
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