Triton x 100, by Promega - Product details

1

Decellularization of Bovine Bone Blocks

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Decellularization of bovine bone blocks started with a wash in PBS plus. Then, four cycles of thermal shock, each comprising a step at 121°C for 20 min, followed by freezing in liquid nitrogen (-196°C) for 16 h, were carried out. During these passages, bone blocks were immersed in bidistilled water (ddH₂O); the solution was changed at every cycle. Cellular debris was then removed by washing bone blocks in 1% Triton X-100 (Promega, Madison, WI, USA) for 8 h, followed by a second wash in 0.1% Triton X-100 for 16 h. Triton X-100 was dissolved in ddH₂O. To remove residual detergent, bone samples were washed two times in ddH₂O for 24 h. All these steps were conducted under continuous shaking at room temperature (RT) with the rotatory shaker VDRL 711 (Asal Srl, Cernusco s/N, Milan, Italy).
At this point, decellularized bone blocks were reduced to granules with dimensions of 0.25–1 mm using Cutting Mill SM 300 (Retsch GmbH, Haan, Germany), then divided into four groups and processed as described in Table 1.

Gardin C., Ricci S., Ferroni L., Guazzo R., Sbricoli L., De Benedictis G., Finotti L., Isola M., Bressan E, & Zavan B. (2015). Decellularization and Delipidation Protocols of Bovine Bone and Pericardium for Bone Grafting and Guided Bone Regeneration Procedures. PLoS ONE, 10(7), e0132344.

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2

Immunofluorescence Staining Protocol

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Cells were fixed using paraformaldehyde 4% in PBS (USB Corporation) for 15 min at room temperature. After washing, cells were permeabilized with 0.1% Triton X-100 (Promega) diluted in dPBS for 15 min. After blocking with 2% of BSA for 4 h, primary antibodies (described below) were incubated overnight at 4°C, and next day secondary antibodies (Life Technologies) were added and kept for 1 h at room temperature. Cells were washed and nucleus was stained with DAPI. Slides were mounted using Pro-long Gold antifade reagent (Invitrogen) (Supplementary Table S12).

Rosa-Fernandes L., Cugola F.R., Russo F.B., Kawahara R., de Melo Freire C.C., Leite P.E., Bassi Stern A.C., Angeli C.B., de Oliveira D.B., Melo S.R., Zanotto P.M., Durigon E.L., Larsen M.R., Beltrão-Braga P.C, & Palmisano G. (2019). Zika Virus Impairs Neurogenesis and Synaptogenesis Pathways in Human Neural Stem Cells and Neurons. Frontiers in Cellular Neuroscience, 13, 64.

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3

Immunolabeling Protocol for Zika Virus

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Cells were fixed using paraformaldehyde, 4% in PBS, for 15 minutes at room temperature. After washing, the cells were permeabilized with 0.1% Triton X-100 (Promega) diluted in PBS for 15 minutes. After blocking with 2% of BSA (Sigma-Aldrich) for 4 hours, primary antibodies directed against the following were added: anti-ZIKV (polyclonal mouse, Institute Pasteur in Dakar, 1:80), anti-Flavivirus D1-4G2-4-15 (polyclonal mouse, Millipore, 1:100), 1:50, anti-MAP2 (chicken, Abcam ab5392, 1:200), anti-cleaved caspase-3 (rabbit, Cell Signaling #9661, 1:400), anti-Sox2 (mouse, Abcam ab97959), anti-GFAP (rabbit, Abcam, 1:500) and anti-Mushashi1 (rabbit, Abcam, 1:1000) (Supplementary Table 2). The cells were incubated overnight at 4°C. Secondary antibodies were added for a one-hour incubation at room temperature, being the following ones: anti-mouse Alexa Fluor 488, anti-chicken Alexa Fluor 647, anti-rat Alexa Fluor 555 and anti-rabbit Alexa Fluor 555 (Invitrogen). The nuclei were stained with DAPI (Invitrogen, 1:10,000) diluted in a PBS 1x solution for 5 minutes and mounted with DPX (Sigma). Images were acquired with Nikon Eclipse 80i. Analysis of data was performed using software NIS Elements 3.22 (Tokyo, Japan).

Cugola F.R., Fernandes I.R., Russo F.B., Freitas B.C., Dias J.L., Guimarães K.P., Benazzato C., Almeida N., Pignatari G.C., Romero S., Polonio C.M., Cunha I., Freitas C.L., Brandão W.N., Rossato C., Andrade D.G., Faria D.D., Garcez A.T., Buchpigel C.A., Braconi C.T., Mendes E., Sall A.A., Zanotto P.M., Peron J.P., Muotri A.R, & Beltrão-Braga P.C. (2016). The Brazilian Zika virus strain causes birth defects in experimental models. Nature, 534(7606), 267-271.

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4

Immunofluorescence Staining of FFPE Tissues

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Immunofluorescence was performed as described previously [103 (link)]. The formalin fixed, paraffin embedded (FFPE) tissue sections were wax removed with Xylene and incubated in Target retrieval solution pH 9.0 (Dako, Glostrup, Denmark) for 10 minutes at room temperature prior to incubating for 15 minutes at 110°C. The sections or cell samples were washed in PBS and fixed in 4% paraformaldehyde (PFA) in PBS for 10 minutes at room temperature. Cells were permeabilised in PBS with 0.1% Triton X-100 (Promega) for 30 minutes, then washed in PBS. The sections or cells were blocked in 5% normal goat serum in PBS for 1 hour prior to incubation of the primary antibodies overnight. The antibodies used were listed in S3 Table. Antigen antibody complexes were visualised with anti-mouse Alexa 488- or 594-conjugated antibody (Thermo Fisher Scientific) or Immpress anti-mouse/rabbit coupled with tyramide amplification kit (PerkinElmer, Inc). Nuclei were counterstained with DAPI.

Yin W., Egawa N., Zheng K., Griffin H., Tian P., Aiyenuro A., Bornstein J, & Doorbar J. (2023). HPV E6 inhibits E6AP to regulate epithelial homeostasis by modulating keratinocyte differentiation commitment and YAP1 activation. PLOS Pathogens, 19(6), e1011464.

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5

Immunofluorescence and Immunohistochemistry of MmuPV1

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Immunofluorescence and immunohistochemistry were performed as previously described (58 (link)). The formalin-fixed, paraffin-embedded tissue sections were incubated in Target retrieval solution (pH 9) (Dako, Glostrup, Denmark) for 10 min at room temperature prior to incubation for 15 min at 110°C. The cells were washed in PBS and fixed in 4% paraformaldehyde (PFA) in PBS for 10 min at room temperature. The cells were permeabilized in PBS with 0.1% Triton X-100 (Promega) for 30 min, then washed in PBS. The sections and cells were blocked in 10% normal goat serum in PBS for 1 h prior to incubation with the primary antibodies. The antibodies used were an in-house rabbit anti-MmuPV1 E4 and L1 monoclonal antibodies (29 (link)), mouse anti-DE-K10 (Invitrogen, CA), rat anti-BrdU, and rabbit anti-cytokeratin 17 (Abcam, Cambridge, United Kingdom). Antigen-antibody complexes were visualized with an anti-mouse Alexa Fluor 488- or 594-conjugated antibody (Thermo Fisher Scientific). Nuclei were visualized with DAPI. BrdU- and K10- positive cells were counted manually.

Saunders-Wood T., Egawa N., Zheng K., Giaretta A., Griffin H.M, & Doorbar J. (2022). Role of E6 in Maintaining the Basal Cell Reservoir during Productive Papillomavirus Infection. Journal of Virology, 96(5), e01181-21.

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6

Immunolabeling Protocol for Zika Virus

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Cells were fixed using paraformaldehyde, 4% in PBS, for 15 minutes at room temperature. After washing, the cells were permeabilized with 0.1% Triton X-100 (Promega) diluted in PBS for 15 minutes. After blocking with 2% of BSA (Sigma-Aldrich) for 4 hours, primary antibodies directed against the following were added: anti-ZIKV (polyclonal mouse, Institute Pasteur in Dakar, 1:80), anti-Flavivirus D1-4G2-4-15 (polyclonal mouse, Millipore, 1:100), 1:50, anti-MAP2 (chicken, Abcam ab5392, 1:200), anti-cleaved caspase-3 (rabbit, Cell Signaling #9661, 1:400), anti-Sox2 (mouse, Abcam ab97959), anti-GFAP (rabbit, Abcam, 1:500) and anti-Mushashi1 (rabbit, Abcam, 1:1000) (Supplementary Table 2). The cells were incubated overnight at 4°C. Secondary antibodies were added for a one-hour incubation at room temperature, being the following ones: anti-mouse Alexa Fluor 488, anti-chicken Alexa Fluor 647, anti-rat Alexa Fluor 555 and anti-rabbit Alexa Fluor 555 (Invitrogen). The nuclei were stained with DAPI (Invitrogen, 1:10,000) diluted in a PBS 1x solution for 5 minutes and mounted with DPX (Sigma). Images were acquired with Nikon Eclipse 80i. Analysis of data was performed using software NIS Elements 3.22 (Tokyo, Japan).

Cugola F.R., Fernandes I.R., Russo F.B., Freitas B.C., Dias J.L., Guimarães K.P., Benazzato C., Almeida N., Pignatari G.C., Romero S., Polonio C.M., Cunha I., Freitas C.L., Brandão W.N., Rossato C., Andrade D.G., Faria D.D., Garcez A.T., Buchpigel C.A., Braconi C.T., Mendes E., Sall A.A., Zanotto P.M., Peron J.P., Muotri A.R, & Beltrão-Braga P.C. (2016). The Brazilian Zika virus strain causes birth defects in experimental models. Nature, 534(7606), 267-271.

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7

Quantifying Keratin10, p53, and MCM in Cells

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2.4x10⁴ cells were seeded in each well of a 4-well culture slide (Falcon) and cultured for 3 and 7 days. The cells were washed in PBS and fixed in 4% paraformaldehyde (PFA) in PBS for 30 min. at room temperature. The cells were permeabilised in PBS with 0.1% Triton X-100 (Promega) for 30 min., then washed in PBS. The cells were blocked in 10% normal goat serum (Cell Signaling Technology) in PBS for 1 hour. The antibodies used were anti-Keratin10 antibody (dilution 1:200, Thermo Fisher Scientific), anti-p53 (DO-1) antibody (dilution 1:300, Santa Cruz), anti-MCM antibody (dilution 1:100, Abcam) an anti-mouse Alexa 594-conjugated antibody (dilution 1:150, Thermo Fisher Scientific). For p53 and MCM, the signal was amplified using a Tyramide Signal Amplification Kit (Perkin-Elmer), according to the manufacturer's instructions. Finally, the cells were mounted in mounting medium (Agar Scientific) for visualization. The number of cells with positive signal were quantified using ilastik software.

Murakami I., Egawa N., Griffin H., Yin W., Kranjec C., Nakahara T., Kiyono T, & Doorbar J. (2019). Roles for E1-independent replication and E6-mediated p53 degradation during low-risk and high-risk human papillomavirus genome maintenance. PLoS Pathogens, 15(5), e1007755.

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8

Quantifying Infiltrating Mononuclear Cells

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The infiltration of mononuclear cells was quantitated by measuring the levels of the lysosomal enzyme N-acetyl-β-D-glucosaminidase (NAG) present in high levels in activated macrophages. Following the Hb measurements the remaining pellets were weighed, homogenized in sodium chloride (NaCl) solution (0.9% w/v) containing 0.1% v/v Triton X-100 (Promega; USA) and centrifuged (3,000 × g; 10 min at 4 °C). Supernatants were incubated for 30 min with an equal volume of 2.24 mM p-nitrophenyl-N-acetyl-beta-D-glucosaminide (Sigma; USA) prepared in citrate/sodium phosphate buffer (0.1 M citric acid, 0.1 M Na₂HPO₄; pH4.5). The reaction was stopped by the addition of glycine buffer (0.8 M glycine, 0.8 M NaCl, 0.8 M NaOH; pH 10.6). Hydrolysis of the substrate was determined by measuring the absorbance at 400 nm. NAG activity was expressed as nmol of p-nitrophenol per mg wet tissue.

Liu D., Andrade S.P., Castro P.R., Treacy J., Ashworth J, & Slevin M. (2016). Low Concentration of Sodium Butyrate from Ultrabraid+NaBu suture, Promotes Angiogenesis and Tissue Remodelling in Tendon-bones Injury. Scientific Reports, 6, 34649.

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9

Decellularization of Whole Liver

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The livers were thawed at room temperature. A feeding tube was inserted into the portal vein and connected to a pump for perfusion with solutions. The first solution used was deionizedwater administered at a rate of 200 mL/min for 1 hour. The liver was then perfused with 1% Triton X-100 (Amresco, Solon, OH, USA) at a rate of 200 mL/min for 3 h and then with 1% SDS (Promega, San Luis Obispo, CA, USA) at the same rate for 6 h. This was followed by 3 h of perfusion with 1% Triton X-100 to remove residual SDS. Subsequently, the liver was washed with 20 L of distilled water to remove residual detergent, followed by infusion of 40 L of PBS at 200 mL/min.

Wang Y., Bao J., Wu X., Wu Q., Li Y., Zhou Y., Li L, & Bu H. (2016). Genipin crosslinking reduced the immunogenicity of xenogeneic decellularized porcine whole-liver matrices through regulation of immune cell proliferation and polarization. Scientific Reports, 6, 24779.

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10

Immunofluorescence Staining of OPN

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Transfected cells grown on glass coverslips were fixed for 30 min in 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 (Promega) in PBS buffered saline for 30 min. After washing with TBS-0.1% Triton X-100 (TBSTx), nonspecific binding sites were blocked with TBSTx-5% BSA (Roche) for 60 min. Then cells were applied sequentially with a 1:100 dilution of rabbit polyclonal antibody anti-OPN (Abcam) at 4°C overnight and a 1:200 dilution of Cy3-conjugated goat anti-rabbit IgG (Beyotime) at room temperature for 60 min in the dark. For negative control, PBS was added instead of primary antibody. Further, immunofluorescence was visualized under a laser-scanning confocal microscope (Zeiss, Oberkochen, Germany).

Xu C., Li H., Yin M., Yang T., An L, & Yang G. (2017). Osteopontin is involved in TLR4 pathway contributing to ovarian cancer cell proliferation and metastasis. Oncotarget, 8(58), 98394-98404.

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Triton x 100

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59 protocols using triton x 100

Decellularization of Bovine Bone Blocks

Immunofluorescence Staining Protocol

Immunolabeling Protocol for Zika Virus

Immunofluorescence Staining of FFPE Tissues

Immunofluorescence and Immunohistochemistry of MmuPV1

Immunolabeling Protocol for Zika Virus

Quantifying Keratin10, p53, and MCM in Cells

Quantifying Infiltrating Mononuclear Cells

Decellularization of Whole Liver

Immunofluorescence Staining of OPN

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