Primary antibodies used were: mouse anti-Wg [1:40, Developmental Studies Hybridoma Bank (DSHB), 4D4], mouse anti-βGal (1:50, DSHB, 40-1a), mouse anti-Mmp1 (1:10, DSHB, 3A6B4/5H7B11/3B8D12 were mixed in equal amounts), mouse anti-Dlg (1:200, DSHB, 4F3) and rabbit anti-cleaved Caspase-3 (1:500, Cell Signaling, 9661S). Samples were dissected in PBS, fixed in 4% formaldehyde for 20 min, washed for 3×10 min in PBX (1% Triton X-100 in PBS), blocked in PBX with 2% BSA (BBX) for 30 min, and incubated in BBX with primary antibody at 4°C overnight. Samples were then washed 4×30 min with BBX to remove primary antibody, and incubated in 200 µl BBX with 1 µl secondary antibody for 2 h. Alexa Fluor 555 series secondary antibodies from Thermo Fisher Scientific were used. After washing for 3×10 min in PBX (including DAPI for 1 wash), samples were mounted on glass slides. Images were taken with a Leica SP8 microscope. Image analysis was performed with Fiji software. Whole larva images were taken with a Leica Fluorescence Stereomicroscope.
Mouse anti wg
Manufactured by Developmental Studies Hybridoma Bank
Sourced in United States
Mouse anti-Wg is an antibody product produced by the Developmental Studies Hybridoma Bank. It is designed to detect the Wg (Wingless) protein in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Rat anti elav, by Developmental Studies Hybridoma Bank (4 mentions)
Donkey anti rabbit igg, by Jackson ImmunoResearch (3 mentions)
Donkey anti mouse igg, by Jackson ImmunoResearch (3 mentions)
Mouse anti ph3, by Cell Signaling Technology (3 mentions)
Vectashield, by Vector Laboratories (3 mentions)
Goat anti rat igg, by Jackson ImmunoResearch (2 mentions)
Fluoview 1000 laser scanning confocal microscope, by Olympus (2 mentions)
Rabbit anti dcp 1, by Cell Signaling Technology (2 mentions)
Anti rabbit 647, by Thermo Fisher Scientific (2 mentions)
Mouse anti nubbin, by Developmental Studies Hybridoma Bank (2 mentions)
Mouse anti mmp1 c terminus, by Developmental Studies Hybridoma Bank (2 mentions)
Mouse anti lacz, by Developmental Studies Hybridoma Bank (2 mentions)
Anti rat 647, by Thermo Fisher Scientific (2 mentions)
Anti mouse 555, by Thermo Fisher Scientific (2 mentions)
Chick anti gfp, by Abcam (2 mentions)
Axio imager m2, by Zeiss (2 mentions)
Rat anti de cadherin, by Developmental Studies Hybridoma Bank (2 mentions)
Axioplan microscope, by Zeiss (2 mentions)
Fluorescence stereomicroscope, by Leica camera (1 mentions)
Sp8 microscope, by Leica camera (1 mentions)
16 protocols using mouse anti wg
1
Immunostaining of Drosophila Larval Tissues
2
Immunolabeling of Drosophila Brain Samples
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Third-instar larval brains were dissected in phosphate-buffered saline (PBS), fixed in 4% formaldehyde for 30 min, washed in PBS+0.1 or 0.3% Triton X-100 (PBT), and blocked in PBT+5% BSA.
Antibodies used were as follows: mouse anti-Wg [Developmental Studies Hybridoma Bank (DSHB) 4D4 1:50 (Brook and Cohen, 1996 (link))], mouse anti-Repo [DSHB 8D12 1:50 (Stork et al., 2014 (link))], mouse anti-Fz1 [DSHB 1C11 1:50 (Park et al., 1994 (link))], mouse anti-Cyt-Arm [DSHB N27A1 1:50 (Riggleman et al., 1990 (link))], mouse anti-MMP1 [DSHB 5H7B11, 3A6B4, 3B8D12, 1:50 (Page-McCaw et al., 2003 (link))], rabbit anti-MMP2 [1:500, (Dear et al., 2016 (link))], guinea pig anti-Grnd [1:250 (Andersen et al., 2015 (link))], mouse anti-β-galactosidase [Sigma-Aldrich G4644, 1:500 (Portela et al., 2019 (link))], mouse anti-GFP [Invitrogen A11120, 1:500 (Djiane et al., 2005 (link))], mouse anti-bruchpilot [DSHB nc82, 1:20 (Wagh et al., 2006 (link))], Rabbit anti-Hrp [Jackson ImmunoResearch 111-035-144, 1:400 (Stork et al., 2014 (link))].
Secondary antibodies: anti-mouse Alexa 488, 568, 647, anti-rabbit Alexa 488, 568, 647 (Thermo Fisher Scientific, 1:500). DNA was stained with 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI, 1 µM).
Antibodies used were as follows: mouse anti-Wg [Developmental Studies Hybridoma Bank (DSHB) 4D4 1:50 (Brook and Cohen, 1996 (link))], mouse anti-Repo [DSHB 8D12 1:50 (Stork et al., 2014 (link))], mouse anti-Fz1 [DSHB 1C11 1:50 (Park et al., 1994 (link))], mouse anti-Cyt-Arm [DSHB N27A1 1:50 (Riggleman et al., 1990 (link))], mouse anti-MMP1 [DSHB 5H7B11, 3A6B4, 3B8D12, 1:50 (Page-McCaw et al., 2003 (link))], rabbit anti-MMP2 [1:500, (Dear et al., 2016 (link))], guinea pig anti-Grnd [1:250 (Andersen et al., 2015 (link))], mouse anti-β-galactosidase [Sigma-Aldrich G4644, 1:500 (Portela et al., 2019 (link))], mouse anti-GFP [Invitrogen A11120, 1:500 (Djiane et al., 2005 (link))], mouse anti-bruchpilot [DSHB nc82, 1:20 (Wagh et al., 2006 (link))], Rabbit anti-Hrp [Jackson ImmunoResearch 111-035-144, 1:400 (Stork et al., 2014 (link))].
Secondary antibodies: anti-mouse Alexa 488, 568, 647, anti-rabbit Alexa 488, 568, 647 (Thermo Fisher Scientific, 1:500). DNA was stained with 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI, 1 µM).
3
Immunostaining of Drosophila Imaginal Discs
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Imaginal discs were dissected from first-, second-, and third-instar larvae in 1XPBS (Phosphate Buffered Saline) and were fixed in 4% para-formaldehyde for 20 minutes. Imaginal discs were washed in PBS after fixation and stained following the standard protocol [47 (link)–49 (link)]. Antibodies used were mouse anti-Wg (1:50) (Developmental Studies Hybridoma Bank), mouse anti-Dlg (1:100), mouse anti β-gal (1:100), rabbit anti-Dlg (1:200; a gift from K. Cho), rat anti Elav (1:100). Secondary antibodies (Jackson Laboratories) used in this study were goat anti-rat IgG conjugated with Cy5 (1:250), donkey anti-rabbit IgG conjugated to Cy3 (1:250), donkey anti-mouse IgG conjugated to FITC (1:300), and donkey anti-mouse IgG conjugated to Cy3 (1:200). The imaginal discs were mounted on slides in Vectashield mountant (Vector Laboratories). Immunofluorescent images were obtained using the Olympus Fluoview 1000 Laser Scanning Confocal Microscope[50 ]. The confocal images were processed using the Photoshop CS6 software.
4
Immunofluorescent Staining of Larval Tissue
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Larvae were dissected in 1 x PBS followed by a 20-min fix in 4% paraformaldehyde in PBS. After 3 washes in 0.1% PBST (1 x PBS + 0.1%Triton-X), larvae were washed in 0.3% PBST and then blocked in 0.1% PBST with 5% normal goat serum (NGS) for 30 min. Primary staining was done overnight at 4°C, whereas secondary staining was done for 4 h at room temperature. The following primary antibodies were obtained from the Developmental Studies Hybridoma Bank: mouse anti-Nubbin (1:25), mouse anti-Wg (1:100), mouse anti-Mmp1 C-terminus (1:100), mouse anti-Mmp1 catalytic domain (1:100), mouse anti-LacZ (1:100), and rat anti-DE-cadherin (1:100). Rabbit anti-DCP1 (1:1000), and mouse anti-PH3 (1:400) were obtained from Cell Signaling Technologies, and chick anti-GFP (1:2000) was obtained from Abcam. The secondary anti-Chick 488 antibody was also obtained from Abcam. Other secondary antibodies, anti-rabbit 647, anti-rat 647, and anti-mouse 555 were obtained from Invitrogen. All secondary fluorophore-conjugated antibodies were used at 1:500. Images were obtained on a Zeiss AxioImager.M2 with ApoTome. For each experiment at least 15 discs were analyzed prior to choosing a representative image. Images were processed using Affinity Photo and Affinity Designer.
5
Immunohistochemistry Protocol for Drosophila
6
Immunostaining of Drosophila Eye-Antennal Discs
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Eye–antennal imaginal discs were dissected from wandering third-instar larvae in 1× PBS and stained following standard protocol (54 (link), 58 (link)). Primary antibodies used were rabbit anti-β-GAL (1:100; Cappel); rat anti-Elav (1:100), mouse anti-Wg (1:100; Developmental Studies Hybridoma Bank, DSHB), mouse anti-Disc large (1:100; DSHB); rabbit anti-Ey (1:200, a gift from Uwe Walldorf and Patrick Callaerts), mouse anti-Eya (1:100; DSHB), mouse anti-Dac (1:100; DSHB), and anti-Rabbit Dve (1:200). Secondary antibodies (Jackson Laboratories) used consisted of donkey anti-mouse IgG conjugated with Cy3 (1:250), donkey anti-rabbit IgG conjugated to Cy3 (1:250), and goat anti-rat IgG conjugated with Cy5 (1:200). The tissues were mounted in Vectashield (Vector Labs), and all the immunofluorescence images were captured using the Olympus FluoView 1000 Laser Scanning Confocal Microscope.
7
Eye-Antenna Disc Immunostaining Protocol
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Eye-antenna discs were dissected from wandering third instar larvae and stained following the standard protocol (Singh et al., 2006) . The eye imaginal discs were dissected in cold 1X solution of Phosphate Buffered Saline (PBS) and fixed using 4% pformaldehyde (Electron Microscopy Sciences, Catalog no. 15710). Fixed tissues were washed using PBS-T [0.2% Triton X-100 (Sigma, Catalog no. T8787) in 1XPBS] and immunostained using primary antibodies: mouse anti-Phospho histone-3 (1:200) (Sigma-Aldrich); Rabbit anti-Drosophila caspase-1 (DCP-1) (1:100) (Sigma-Aldrich); rat anti-Elav (1:100) (Developmental Studies Hybridoma Bank); mouse anti-Wg (1:100) (Developmental Studies Hybridoma Bank), and goat anti-Hth (a gift from H. Sun and R.
8
Immunostaining of Drosophila Eye-Antennal Discs
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Eye-antennal discs of wandering third instar larvae were dissected in 1 × phosphate buffered saline (PBS), fixed in 4% paraformaldehyde in PBS (fixative) for 20 min and washed in PBST (three times). The tissues were stained with a combination of antibodies following the standard protocol39 (link). Primary antibodies used were rabbit anti-β-GAL (1:100; Cappel); rat anti-Elav (1:100), mouse anti-Wg (1:100; Developmental Studies Hybridoma Bank, DSHB), mouse anti- Dlg (1:100); mouse anti-Eya (1:100; DSHB), mouse anti-Dac (1:100; DSHB), goat anti-Hth (1:200; Santa Cruz), mouse anti-Sca (1:100), goat anti-Ato (1:50), rabbit anti-Dcp1 (1:150, Santa Cruz), mouse anti-pH3 (1:300, Cell Signaling). The discs were washed in PBST thrice for 10 min. Secondary antibodies used were donkey anti-rat IgG conjugated to Cy5 (1:250), donkey anti-rabbit IgG conjugated to Cy3 (1:300) or goat anti-mouse IgG conjugated to FITC (1:200) (Jackson Laboratories). The discs were mounted in Vectashield and photo-documented on a Fluoview 3000 Laser Scanning Confocal Microscope70 (link). We took the images at 20 × magnification unless stated otherwise. We analyzed and prepared the final figures with images using Adobe Photoshop CS6 software.
9
Immunostaining of Drosophila Eye-Antennal Discs
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Eye-antennal discs from wandering third instar larvae were dissected, and fixed in 4% paraformaldehyde in Phosphate Buffered Saline (PBS), and stained following the protocol85 (link)–87 (link). The primary antibodies used were rabbit anti-Dlg (1:200; a gift from K. Cho), mouse anti-Wg [1:50,Developmental Studies Hybridoma Bank,(DSHB)], rat anti-Elav (1:50; DSHB), mouse anti-Dlg (1:100; DSHB), mouse anti-22C10 (1:100; DSHB), mouse anti-Chaoptin (MAb24B10) (1:100; DSHB88 ), mouse anti-Ey (1:100, DSHB), mouse anti-Eya (1:100, DSHB), mouse anti-Dac (1:100, DSHB), mouse anti-β-galactosidase (1:100; DSHB), rabbit anti-β-galactosidase (1:200) (Cappel), and mouse anti-GFP (1:100, GFP-G1, DSHB). Secondary antibodies (Jackson Laboratories) used consisted of donkey anti-rabbit IgG conjugated with FITC (1:200), donkey anti-mouse IgG conjugated with Cy3 (1:250), and goat anti-rat IgG conjugated with Cy5 (1:250). The tissues were mounted in Vectashield (Vector labs) and all immunofluorescence images were captured using the Laser Scanning Confocal Microscopy89 (Olympus Fluoview 1000). All images were taken at 20X magnification unless stated otherwise. The final images and figures were prepared using Adobe Photoshop CS6 software.
10
Immunohistochemical Analysis of Drosophila Larvae
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Larvae were dissected in PBS followed by a 20-minute fix in 4% paraformaldehyde in PBS. After 3 washes in 0.1% PBST (Triton-X), larvae were washed in 0.3% PBST and then blocked in 0.1% PBST with 5% NGS for 30 minutes. Primary staining was done overnight at 4 o C, while secondary staining was done for 4 hours at room temperature.
The following primary antibodies were obtained from the Developmental Studies Hybridoma Bank: mouse anti-Nubbin (1:25), mouse anti-Wg (1:100), mouse anti-Mmp1 C-terminus (1:100), mouse anti-Mmp1 catalytic domain (1:100), mouse anti-LacZ (1:100), and rat anti-DE-Cadherin (1:100). Rabbit anti-Dcp1 (1:1000) and mouse anti-PH3 (1:400) was obtained from Cell Signaling Technologies, and chick anti-GFP (1:2000) was obtained from Abcam. The secondary anti-Chick 488 antibody was also obtained from Abcam. Other secondary antibodies, anti-rabbit 647, anti-rat 647, and anti-mouse 555 were obtained from Invitrogen. All secondary fluorophore-conjugated antibodies were used at 1:500. Images were obtained on a Zeiss AxioImager.M2 with ApoTome. For each experiment at least 15 discs were analyzed prior to choosing a representative image.
Images were processed using Affinity Photo and Affinity Designer.
The following primary antibodies were obtained from the Developmental Studies Hybridoma Bank: mouse anti-Nubbin (1:25), mouse anti-Wg (1:100), mouse anti-Mmp1 C-terminus (1:100), mouse anti-Mmp1 catalytic domain (1:100), mouse anti-LacZ (1:100), and rat anti-DE-Cadherin (1:100). Rabbit anti-Dcp1 (1:1000) and mouse anti-PH3 (1:400) was obtained from Cell Signaling Technologies, and chick anti-GFP (1:2000) was obtained from Abcam. The secondary anti-Chick 488 antibody was also obtained from Abcam. Other secondary antibodies, anti-rabbit 647, anti-rat 647, and anti-mouse 555 were obtained from Invitrogen. All secondary fluorophore-conjugated antibodies were used at 1:500. Images were obtained on a Zeiss AxioImager.M2 with ApoTome. For each experiment at least 15 discs were analyzed prior to choosing a representative image.
Images were processed using Affinity Photo and Affinity Designer.
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