Tissues and tumors were fixed in 10% formalin and cells from culture were fixed in 4% paraformaldehyde and stained as described [6 , 14 (link), 15 (link)]. Primary antibodies were as follows: anti-E-cadherin R&D AF748 (1:100); anti-N-Cadherin Abcam ab98952 (1:100); anti-CD3 BD Pharmingen 550275 (1:100). The secondary antibodies were as follows: Donkey Alexa 488-conjugated IgG or Cy3-conjugated IgG. Nuclei were stained with DAPI or Hoechst 33258 (Invitrogen). Images were acquired with a confocal Leica TCS SP5 (Leica), Carl Zeiss upright Apotome Axio Imager Z1 or Cytaion5 (Biotek).
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Immunofluorescence Staining of Tissues and Cells
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Anti cd3
Anti d
Antibodies
Cadherin
Cells culture
Dapi
Donkey
Formalin
Hoechst 33258
Nuclei
Paraformaldehyde
Tissues
Tumors
Corresponding Organization :
Other organizations : Brown Foundation, The University of Texas at Austin
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Variable analysis
independent variables
- Fixation method of tissues and tumors (10% formalin)
- Fixation method of cells from culture (4% paraformaldehyde)
- Expression of E-cadherin
- Expression of N-cadherin
- Expression of CD3
- Staining protocol as described in references [6], [14], and [15]
- Primary antibodies: anti-E-cadherin (1:100), anti-N-cadherin (1:100), anti-CD3 (1:100)
- Secondary antibodies: Donkey Alexa 488-conjugated IgG or Cy3-conjugated IgG
- Nuclei staining with DAPI or Hoechst 33258
- Imaging equipment: confocal Leica TCS SP5, Carl Zeiss upright Apotome Axio Imager Z1, or Cytaion5 (Biotek)
- Positive controls not explicitly mentioned
- Negative controls not explicitly mentioned
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