Formalin-fixed paraffin-embedded tissue sections 5 μm thick were incubated in a 60° oven for 1 h and hydrated through a series steps of xylene and ethanol baths to water. Deparaffinized sections were boiled in antigen retrieval solution (100X citrate buffer, pH 6.0, Abcam, Cambridge, MA) for 10 min and cooled down for 20 min. Slides were then washed with hot tap water for 1 min and PBS twice for 3 min each time. After blocking in 5% normal goat serum diluted in PBS, the sections were incubated with anti-ACE2 antibody (ab108252, 1: 100, Abcam, Cambridge, MA), anti-TMPRSS2 antibody (ab92323, 1: 1000, Abcam) and the isotype- and concentration-matched normal IgG control (ab172730, Abcam) overnight at 4 °C. After 3 washes with PBST (PBS plus 0.1% Tween-20), the sections were incubated with the secondary biotinylated goat anti-rabbit IgG (1:2000) for 1 h at room temperature. The sections were then detected by alkaline phosphatase detection system (Vectastain ABC-AP kit, Vector laboratories, Burlingame, CA) and a blue reaction product was produced by incubating sections with alkaline substrate (Vector blue AP substrate kit III, Vector laboratories) for 20–40 min. The sections were also counterstained with nuclear fast red (Vector laboratories) and mounted with mounting medium (H-5501, Vector laboratories), and examined under a light microscope.
H 5501
Manufactured by Vector Laboratories
Sourced in United States
The H-5501 is a general-purpose laboratory centrifuge designed for a variety of applications. It features a compact and durable construction, with a maximum speed of 5,000 rpm and a maximum relative centrifugal force (RCF) of 3,421 g. The centrifuge accommodates a range of sample volumes and tube sizes, making it suitable for tasks such as sample preparation, cell separation, and other common laboratory procedures.
Automatically generated - may contain errors
Lab products found in correlation
Ab92323, by Abcam (2 mentions)
Ab172730, by Abcam (2 mentions)
Vector blue ap substrate kit 3, by Vector Laboratories (2 mentions)
Nuclear fast red, by Vector Laboratories (2 mentions)
Vectastain abc ap kit, by Vector Laboratories (2 mentions)
Ab108252, by Abcam (2 mentions)
Axioplan 2, by Zeiss (2 mentions)
Fluorescence microscopy, by Vector Laboratories (1 mentions)
Ab93678, by Abcam (1 mentions)
Ab7481, by Abcam (1 mentions)
Er tracker red dye, by Thermo Fisher Scientific (1 mentions)
Gtx100554, by GeneTex (1 mentions)
7 protocols using h 5501
1
Immunohistochemical Analysis of ACE2 and TMPRSS2 Expression
2
Immunofluorescence Staining of Cellular Markers
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Cells were cultured on coverslips, washed with PBS, fixed with 4% parafprmaldehydefor 30 min at RT, and then permeabilized in 0.1% Triton X-100/PBS at RT for 10 min. After treatment with blocking solution (anti-Human antibody diluted 1:500 in PBS) for 1 h, cells were incubated with anti-Lamin A/C (1:500), p16 (1:300), and H3K9me3 (1:300) in blocking solution overnight at 4 °C. Finally, cells were incubated with fluorescein isothiocyanate (FITC) and Rhodamine-conjugated secondary antibodies at 4 °C for 6 h. The nucleus was stained with DAPI (4, 6-diamidino-2-phenylindole) for 10 min. After cells were washed three times with PBS, coverslips were mounted with mounting solution (H-5501; Vector Laboratories (Burlingame, CA, USA) and analyzed by fluorescence microscopy (Zeiss, Axioplan 2) at × 400 magnification.
3
Immunofluorescence Staining of Cellular Markers
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Cells were cultured on coverslips, washed with PBS, fixed with 4% paraformaldehyde (PFA) for 30 min at RT, and then permeabilized with 0.2% Triton X-100 at RT for 5 min. After treatment with blocking solution (anti-Human antibody diluted 1:400 in PBS) for 1 h, cells were incubated with anti-lamin A/C (1:400), anti-progerin (1:100), Ki67 (1:200), and anti-H3K9me3 (1:200) in blocking buffer overnight at 4 °C. Finally, cells were incubated with fluorescein isothiocyanate and rhodamine-conjugated secondary antibodies at 4 °C for 7 h. Nuclei were stained with DAPI (4, 6-diamidino-2-phenylindole) at RT for 10 min. After cells were washed three times with PBS, coverslips were mounted with mounting solution (H-5501; Vector Laboratories, Burlingame, CA, USA). Immunofluorescence signal was detected with a fluorescence microscopy (Zeiss and Logos).
4
Immunofluorescence Analysis of SOD1 in Cells
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Cells on coverslips were washed with PBS and fixed with 4% PFA for 30 min at room temperature and then permeabilized in 0.1% Triton X-100/PBS for 10 min. After cells were treated with blocking solution (anti-Human Antibody diluted 1:500 in PBS) for 1 h, cells were incubated with anti-pan SOD1 (diluted in 1:400) for overnight at 4 °C. Finally, the cells were incubated with FITC-conjugated secondary antibodies at 4 °C for 6 h. The nucleus was stained with 4, 6-diamidino-2-phenylindole (DAPI) and the endoplasmic reticulum (ER) was stained using ER-Tracker Red dye for 10 min. Cells were washed three times with PBS, then the coverslips were mounted with the mounting solution (H-5501; Vector Laboratories (Burlingame, CA, USA)) and analyzed by fluorescence microscopy (Zeiss).
5
Immunohistochemical Staining for ACE2 and TMPRSS2
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Immunohistochemistry staining was performed as previous described.30 (link) Briefly, deparaffinized sections were boiled in 100 × antigen retrieval buffer (ab93678, Abcam, Cambridge, MA) for 20 minutes. After blocking in 5% normal goat serum diluted in PBS, the sections were incubated with anti-ACE2 antibody (ab108252, 1: 100; Abcam, Cambridge, MA, USA), anti-TMPRSS2 antibody (ab92323, 1:2000; Abcam), and isotype- and concentration-matched normal IgG control (ab172730, Abcam) overnight at 4°C. The sections were incubated with secondary biotinylated goat anti-rabbit IgG (1:2000) for one hour at room temperature. ACE2 staining was then detected using the alkaline phosphatase detection system (Vectastain ABC-AP kit, Vector laboratories, Burlingame, CA), and a blue reaction product was produced by incubating sections with alkaline phosphatase substrate (Vector blue AP substrate kit III; Vector Laboratories, Burlingame, CA, USA) for 20 to 30 minutes. The sections were counterstained with nuclear fast red (Vector Laboratories), mounted with mounting medium (H-5501, Vector Laboratories), and examined under a light microscope. For quantitation of ACE2 positive blood vessels, figures at magnification ×10 were taken from four consecutive fields adjacent to the optic nerve. The number of ACE2 positive vessels was quantified by two independent observers in a masked fashion.
6
Immunofluorescence analysis of SOD1 inclusions
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The transfected cells on coverslips were fixed with 4% paraformaldehyde for 30 min at room temperature. The cells were then permeabilized in 0.1% Triton X-100/PBS for 10 min, followed by 1 h of treatment with a blocking solution consisting of normal goat serum (1:200, Abcam, ab7481) in PBS. The pan-SOD1 antibody (1:400, Genetex; GTX100554) solution, diluted in the blocking solution, was added to the cell solution overnight at 4 °C. After washing with PBS, we incubated the cells with Alexa Fluor 488 antibody (Goat anti-Rabbit IgG, 1:500, Thermo Fisher Scientific, #A-11008) at 4 °C for 6 h. The nucleus was stained with 4,6-diamidino-2-phenylindole (DAPI), and the endoplasmic reticulum (ER) was stained using ER-TrackerTM Red dye (Invitrogen) for 10 min. After washing the coverslips three times with PBS, we mounted the coverslips with mounting solution (H-5501; Vector Laboratories) for fluorescence microscopy (Zeiss).
For inclusion positive cells counting, immunofluorescence images with SOD1 antibodies were counted in randomly selected fields. Depending on SOD1 staining, inclusion positive cells were counted based on the strong intensity of SOD1 and expressed as a percentage of total cells counted.
For inclusion positive cells counting, immunofluorescence images with SOD1 antibodies were counted in randomly selected fields. Depending on SOD1 staining, inclusion positive cells were counted based on the strong intensity of SOD1 and expressed as a percentage of total cells counted.
7
Immunofluorescence Staining of Aurora A and γ-Tubulin
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Cells on coverslips were washed with PBS and fixed with 4% PFA for 30 min at room temperature and then permeabilized in 0.1% Triton X-100/PBS for 10 min. After cells were treated with blocking solution (anti-Human Antibody diluted 1:500 in PBS) for 1 hr, cells were incubated with anti-Aurora A (diluted in 1:200), γ-tublin (T6557; Sigma; diluted 1:500 in blocking solution) for overnight at 4°C. Finally, the cells were incubated with FITC and Rhodamine-conjugated secondary antibodies at 4°C for 6 hr. The nucleus was stained with 4, 6-diamidino-2-phenylindole (DAPI) for 10 min. Cells were washed three times with PBS, then the coverslips were mounted with mounting solution (H-5501; Vector Laboratories (Burlingame, CA, USA)) and analyzed by fluorescence microscopy (Zeiss Axioplan2, Oberkochen, Germany), 400x magnification.
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