Alexa fluor 488 goat anti rabbit

In 4 mammosomatotropinomas, confocal laser scanning microscopy (Olympus FV1000D, Japan) was performed using the same primary antibodies (GH/NeuroD1 and PRL/NeuroD1 cocktail). Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 647 goat anti-mouse (Abcam, UK) were used as secondary antibodies. Nuclei were stained with DAPI (appliChem). Details of the confocal laser scanning microscopy method are given in the Supplementary Materials.

NeuN was used to label mature neurons and to reflect the number of neurons in DG. The sections were rewarmed, membrane-ruptured with Triton-X, blocked with goat serum, and incubated with rabbit anti-NeuN antibody (1:1000, Abcam, UK) overnight at 4 °C. After being washed in TBST, the sections were incubated with AlexaFluor^® 488 goat anti-rabbit (1:500, Abcam, UK) at 37 °C for 2 h. After being washed in TBST and DAPI staining, images were captured by a laser confocal microscope (LSM800, ZEISS, Germany). The ratio of NeuN positive cells (%) = NeuN positive cell number/total number of nuclei × 100%.

The tissue samples of spinal sacral nerves and bladders were immersed in OCT, frozen at -20 °C, and cut into 10-μm-thick sections. The nerve and bladder sections were then mounted on glass slides. Next, the slides were treated for 15 min with 0.1% Triton X-100 in PBS, and then blocked for 1 h in 10% normal goat serum (Cayman Chemical, Ann Arbor, MI, USA). After that, the nerve slides were incubated overnight with primary antibodies [rabbit anti-S100 polyclonal antibody (ab11428, 1:200; Abcam, Cambridge, UK) and mouse anti-neurofilament antibody (ab215903, 1:200; Abcam, Cambridge, UK)] at 4 °C, washed with PBS, and incubated with secondary antibodies [Alexa Fluor 488 goat anti-mouse (Abcam, Cambridge, UK) and Alexa Fluor 594 goat anti-rabbit (Abcam, Cambridge, UK)] at room temperature for 2 h. All the bladder slides were incubated overnight with primary antibodies [mouse anti-collagen 1 polyclonal antibody (ab88147, 1:200; Abcam, Cambridge, UK) and rabbit anti-collagen 3 antibody (ab6310, 1:200; Abcam, Cambridge, UK)] at 4 °C, washed with PBS, and incubated with secondary antibodies [Alexa Fluor 594 goat anti-mouse (Abcam, Cambridge, UK) and Alexa Fluor 488 goat anti-rabbit (Abcam, Cambridge, UK)] at room temperature for 2 h. All slides were finally covered with a cover glass. The slides were examined and imaged using a fluorescence microscope (Leica, Wetzlar, Hessen, Germany).

For immunofluorescence staining, kidney sections were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.25% Triton X-100. After blocking in 3% BSA for 60 min, slides were incubated with the first antibody diluted in 1% BSA overnight. After washing with PBS, coverslips were incubated overnight with primary antibodies (Mitofilin, and RIP3) and with the secondary antibodies Alexa Fluor 488 Goat Anti-Rabbit (Abcam Cat# ab150077), and Alexa Fluor 647 Goat Anti-Mouse (Abcam, Cat# ab150119). Images were taken on a Zeiss Axiovert 200M inverted motorized fluorescence microscope (Carl Zeiss Microscope, Jena, Germany).

Dorsal skin tissue samples were collected at various time points following the final UVA irradiation; 1, and 6 h post-irradiation for Nrf2 and its target proteins, respectively; 1 h post-irradiation for oxidative DNA damage; 24 h post-irradiation for MMP-1 and collagen. Tissue sections were washed with PBS for 5 min/time (3 times) and blocked with phosphate-buffered saline (PBS) containing 2% BSA for 30 min. After removing excess blocking buffer, the slides were incubated with Nrf2 Ab (ab31163; Abcam, Cambridge, MA, United States), GST Ab (sc-459; Santa Cruz Biotechnology, Santa Cruz, CA), NQO1 Ab (ab34173; Abcam, Cambridge, MA, United States) (1:50), 8-OHdG [N45.1] Ab (ab48508; Abcam, Cambridge, MA, United States) (1:50), MMP-1 Ab (ab137332; Abcam, Cambridge, MA, United States) (1:50), collagen I (C-18) Ab (sc-8784; Santa Cruz Biotechnology, Santa Cruz, CA) (1:50) for 1 h. The slides were then washed for 5 min/time (3 times) with a PBS solution and incubated for 1 h at room temperature with FITC-conjugated the secondary Ab (green) and with DAPI (blue) to counterstain the nuclei for detection of nuclear Nrf2, the secondary Ab Alexa Fluor 488 goat anti-rabbit (Abcam) for detection of MMP-1 and collagen levels. An inverted fluorescent microscope equipped with a Nikon Intensilight was used for the imaging of IF stainings (20X) which were quantified using ImageJ software.

To examine AAT distribution, MDMs were differentiated on glass slides and fixed in 4% paraformaldehyde for 20 min and permeabilized for 10 min in PBS containing 0.01% Triton X-100. The permeabilized cells were incubated with rabbit anti AAT polyclonal antibody (Abcam, Cambridge) at 1:400 dilution in PBS containing 0.1% Tween 20 for 1 h. After washing with PBS-Tween 20, cells were immunostained with Alexa Fluor488 goat anti-rabbit (Abcam, Cambridge) at 1:500 dilution at room temperature for 1 h. The immunostained cells were mounted on glass slides using VECTASHIELD mounting media with DAPI and examined using a fluorescence microscope (BZ-X700, Keyence, Osaka). For quantification of intracellular AAT, AAT fluorescent intensity and number of cells were measured with BZ software, and the fluorescent intensity was normalized to the cell number. To examine CD36 distribution, MDMs were incubated with mouse anti CD36 monoclonal antibody (ThermoFisher, Waltham) at 1:20 dilution in blocking solution (Invitrogen, Carlsbad) overnight at 4°C. After washing with PBS-Tween 20, cells were immunostained with Alexa Fluor647 goat anti-rabbit (Abcam, Cambridge) at 1:1000 dilution at room temperature for 1 h. For quantification of CD36, ~1000 MDMs were evaluated for each MDM group (n=4).