phosphorylate smad2/3 d27f4 psmad2/3 #8828 antibody  (Cell Signaling Technology Inc)

Journal: STAR protocols

Article Title: Protocol for immunofluorescence detection and quantification of phosphorylated SMAD proteins in human blastocysts.

doi: 10.1016/j.xpro.2025.103849

Figure Lengend Snippet: Figure 1. Immunohistochemistry procedure and confocal microscopy data of phospho-SMAD detection in human blastocysts (A) Representative images illustrating key steps in the immunostaining procedure for phospho-SMAD protein detection. (i) Step 5. Acetone and watch glasses for subsequent incubation should be pre-chilled on ice. Covering watch glasses with cling film prevents the build-up of condensate. (ii) Step 11. Transfer of human embryos into pre-chilled acetone using hand-made glass capillaries fitted with a rubber bulb. (iii) Step 11. Once the human embryo has been placed in acetone, the watch glass is transferred onto dry ice immediately. (iv) Step 12. After incubation in acetone, each human embryo is individually recovered into 0.1% Triton-PBS in 4-well dishes. (B) Confocal microscopy detection of nuclear staining (DAPI) in human blastocysts following acetone treatment. Expanded blastocysts can collapse and become flattened (top row). This does not preclude the confocal microscopy data acquisition. (C) Resulting immunohistochemistry detection of phospho-SMAD protein expression in human blastocysts, and nuclear staining (DAPI). No detection of phospho-SMAD2 at 5 days post fertilization (dpf) (top row), emerging phospho-SMAD2 (middle row), and strong phospho-SMAD2 expression (bottom row). Distinct levels of phospho-SMAD1/5 expression are detectable within the same embryo (bottom panel). Yellow arrow heads indicate examples of nuclei expressing the indicated phospho-SMAD protein. (D) Immunohistochemistry of phospho-SMAD2 and NANOG expression in human blastocysts, processed entirely in parallel. Natural variability between human embryos can result in different levels of background staining. Scale bar 50 μm.

Article Snippet: Furthermore, both antibodies for detection of phosphorylated SMAD2 and SMAD1/5 (Cell Signaling Technology, Cat# 18338 and Cat# B5B10, respectively) were raised in rabbit and cannot be combined in one analysis.

Techniques: Immunohistochemistry, Confocal Microscopy, Immunostaining, Incubation, Staining, Expressing

Journal: STAR protocols

Article Title: Protocol for immunofluorescence detection and quantification of phosphorylated SMAD proteins in human blastocysts.

doi: 10.1016/j.xpro.2025.103849

Figure Lengend Snippet: Figure 4. Manual validation of nuclei with high DAPI-normalized fluorescence intensity (A) Nuclear staining (DAPI) in a hatching human blastocyst (left), the resulting tracked objects (middle) and the overlay onto the original microscopy data (right), to illustrate the fidelity of nuclear segmentation and tracking. (B) Immunohistochemistry detection of NANOG and phospho-SMAD2 expression in the same Z-plane displayed in (A) and overlaid with the tracked nuclei (bottom row). (C) DAPI-normalized phospho-SMAD2 immunofluorescence intensity of nuclei highlighted in and, in (A) and (B). Nuclei 38, 53, and 29 (green) had confirmed phospho-SMAD2 expression, while nucleus 45 (gray) does not display phospho-SMAD2 expression. (D) Bee swarm plot of NANOG and phospho-SMAD2 fluorescence intensity of all nuclei in one analyzed human blastocyst, either raw or DAPI-normalized. Error bars indicate mean +/- SEM. Highlighted are nuclei with manually validated expression of NANOG (red) or phospho-SMAD2 (green). DAPI normalization results in a reduction of false- positive phospho-SMAD2-expressing nuclei amongst the tracked object with highest normalized phospho-SMAD2 immunofluorescence intensity.

Article Snippet: Furthermore, both antibodies for detection of phosphorylated SMAD2 and SMAD1/5 (Cell Signaling Technology, Cat# 18338 and Cat# B5B10, respectively) were raised in rabbit and cannot be combined in one analysis.

Techniques: Biomarker Discovery, Fluorescence, Staining, Microscopy, Immunohistochemistry, Expressing, Immunofluorescence