Donkey anti rat igg

For indirect immunohistochemistry (IHC), 10% neutral-buffered formalin (NBF)-fixed paraffin sections were processed for heat-based antigen unmasking in 10 mM sodium citrate [pH 6.0], with the exception of ATF3, which used 10 mM Tris-HCl, 1 mM EDTA [pH 9.0]. Sections were incubated with antibodies at the following dilutions: 1:200 ARID1A (D2A8U) (12354, Cell Signaling); 1:400 Phospho-S6 (4585, Cell Signaling); 1:100 KRT8 (TROMA1, DHSB); 1:200 Cleaved Caspase-3 (9579, Cell Signaling); 1:400 Ki67 (12202, Cell Signaling); 1:200 ATF3 (GTX37776, GeneTex); 1:200 TP63 (N2C1, GeneTex); 1:200 TP63 (13109, Cell Signaling); 1:100 COL17A1 (ab184996, abcam). The following Biotin-conjugated secondary antibodies were used: donkey anti-rabbit IgG (711-065-152, Jackson Immuno-research Lab) and donkey anti-rat IgG (#705-065-153, Jackson Immuno-research Lab). Secondary antibodies were detected using VECTASTAIN Elite ABC HRP Kit (Vector). Sections for IHC were lightly counter-stained with Hematoxylin QS or Methyl Green (Vector Labs). Routine Hematoxylin and Eosin (H&E) staining of sections was performed by the VARI Histology and Pathology Core. Adjacent sections were used for H&E and IHC marker comparisons as in Fig 8. At least four animals per genotype were assayed for each histological analysis and immunohistochemical marker.

Dopamine (Sigma-Aldrich, catalog# H8502-5G, Schnelldorf, Germany) was dissolved in ddH2O to obtain a stock solution with a concentration of 10 M, and the desired working solutions were then prepared. The primary antibody was anti-GFP (green fluorescent protein) rat monoclonal IgG from Santa Cruz Biotechnology (Heidelberg, Germany), and the secondary antibody was donkey anti-Rat IgG from Jackson Immuno Research Europe (Heidelberg, Germany).

The maxilla was fixed overnight at 4°C in 4% paraformaldehyde/PBS solution, and then washed for 1 week in EDTA 0.5 M/PBS that was changed every other day. The tissue was cryopreserved in 30% sucrose (overnight at 4°C), embedded in OCT, and cryosectioned into 10 µm-thick sections. The cross sections, as well as the separated epithelium, were washed three times in PBS, blocked in a blocking buffer (5% FCS, 0.1% Triton X-100 in PBS) for 1 h at room temperature, and incubated with primary antibodies: goat anti-GAS6 (clone sc-1935, Santa Cruz Biotechnology), rabbit anti-PROS1 (clone 2428718, Millipore), rat anti-CD31 (clone 550274, BioLegend), goat anti-AXL (clone sc-1096, Santa Cruz Biotechnology), and rabbit-Anti-LYVE1 (clone-ab14917, Abcam) overnight at 4°C. Following three washing steps in PBS, the samples were incubated with a secondary antibody: donkey anti-goat IgG, donkey anti-rat IgG, or donkey anti-rabbit IgG (Jackson ImmunoResearch) diluted 1:100 in blocking buffer for 1 h at RT, washed three times, stained with hoechst, and mounted. As a negative staining control, primary antibody was omitted and replaced by blocking buffer. Signals were visualized and digital images were obtained using an Olympus BX51 fluorescent microscope mounted with a DP72 (Olympus) camera.

The following antibodies were used for immunofluorescence (IF) analysis and Western blotting (WB): rat monoclonal anti-α-tubulin (1:800 for IF, 1:1000 for WB; MCA78G, Bio-Rad Laboratories, Hercules, CA, USA), rabbit monoclonal anti-phospho-Akt (Ser473) (1:2000 for WB; D9E, Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal anti-γ-tubulin (1:400 for IF; GTU-88, Sigma-Aldrich), and rabbit polyclonal anti-cleaved caspase-3 (1:500 for IF; 1:500 for WB; Asp175, #9661, Cell Signaling Technology). In terms of secondary antibodies, Alexa Fluor 488- or 555-labeled, donkey anti-rabbit or goat anti-rat IgG (1:400–1:800; Life Technologies, Waltham, MA, USA) was used for IF. Horseradish peroxidase (HRP)-conjugated donkey anti-mouse IgG (1:4000, 715-035-151), donkey anti-rabbit IgG (1:4000, 711-035-152), and donkey anti-rat IgG (1:4000, 712-035-153) antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA) and were used as secondary antibodies for WB.

The primary antibodies used for immunofluorescence (IF) and WB analyses were rabbit monoclonal anti-ALK (WB, 1:500–1000; IF, 1:200; #3333S, Cell Signaling Technology, Danvers, MA, USA), anti-pALK Y1507 (WB, 1:1000; #14678, Cell Signaling Technology) and anti-c-Met (WB, 1:1000; #8198, Cell Signaling Technology); rat monoclonal anti-α-tubulin (WB, 1:1000; IF, 1:800; MCA78G, Bio-Rad Laboratories, Hercules, CA, USA) and anti-HA (WB, 1:1000; IF, 1:400; 3F10, Roche, Basel, Switzerland); goat anti-Lamin B (WB, 1:200–500; sc-6216, Santa Cruz Biotechnology, Dallas, TX, USA); and mouse monoclonal anti-Actin (WB, 1:2000; A3853, MilliporeSigma, Burlington, MA, USA). Secondary antibodies included Alexa Fluor 555-labeled goat anti-rat IgG (1:1000; Life Technologies, Waltham, MA, USA); 488-labeled donkey anti-rabbit and anti-rat IgG (1:800; Life Technologies) for IF. The antibodies used for WB included horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG (1:4000; 711-035-152), donkey anti-rat IgG (1:4000; 712-035-153), donkey anti-mouse IgG (1:4000; 715-305-151) from Jackson Immuno Research (West Grove, PA, USA), and bovine anti-goat (1:4000; sc-2350) from Santa Cruz Biotechnology.

For indirect immunohistochemistry (IHC), 10% neutral-buffered formalin (NBF)-fixed paraffin sections were processed for heat-based antigen unmasking in 10 mM sodium citrate [pH 6.0]. Sections were incubated with antibodies at the following dilutions: 1:200 ARID1A (D2A8U) (12354, Cell Signaling); 1:400 Phospho-S6 (4585, Cell Signaling); 1:100 KRT8 (TROMA1, DHSB); 1:100 EPCAM (G8.8-s, DHSB); 1:400 PGR (SAB5500165, Sigma). TROMA-I antibody was deposited to the DSHB by Brulet, P./Kemler, R. (DSHB Hybridoma Product TROMA-I). EPCAM antibody (G8.8) was deposited to the DSHB by Farr, A.G. (DSHB Hybridoma Product G8.8). Antibody details are listed in Supplementary Table 2. The following Biotin-conjugated secondary antibodies were used: donkey anti-rabbit IgG (711-065-152, Jackson Immuno-research Lab) and donkey anti-rat IgG (#705-065-153, Jackson Immuno-research Lab). Secondary antibodies were detected using VECTASTAIN Elite ABC HRP Kit (Vector). Sections for IHC were lightly counter-stained with Hematoxylin QS or Methyl Green (Vector Labs). Routine Hematoxylin and Eosin (H&E) staining of sections was performed by the Van Andel Research Institute (VARI) Histology and Pathology Core. A VARI animal pathologist reviewed histological tumor assessments.