Protocol detail

Immunostaining and F-actin Visualization

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For immunostaining, we followed standard procedures (Williams et al., 2014 (link)). For F-actin staining, Alexa-coupled Phalloidin (1:200; Invitrogen) was added in PBS/2% Tween-20 for 30 min prior to final washes and mounting. The samples were mounted in Vectashield (Vector)+DAPI, or incubated with the DNA dye TOPRO-3 (1:1000; Molecular Probes) for 10 min and then mounted in Vectashield (Vector).

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Ng B.F., Selvaraj G.K., Santa-Cruz Mateos C., Grosheva I., Alvarez-Garcia I., Martín-Bermudo M.D, & Palacios I.M. (2016). α-Spectrin and integrins act together to regulate actomyosin and columnarization, and to maintain a monolayered follicular epithelium. Development (Cambridge, England), 143(8), 1388-1399.

Publication 2016

Vectashield TOPRO-3

Dapi F actin Molecular probes Phalloidin Tween 20 Vector

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Variable analysis

independent variables

Immunostaining protocol (following standard procedures from Williams et al., 2014)
F-actin staining (using Alexa-coupled Phalloidin)
DNA staining (using TOPRO-3)

dependent variables

Immunostaining signal
F-actin distribution
DNA staining pattern

control variables

PBS/2% Tween-20 buffer
Vectashield mounting medium
Incubation times (30 min for Phalloidin, 10 min for TOPRO-3)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

Based on most similar protocols

For F-actin staining, Alexa-coupled Phalloidin (1:200; Invitrogen) was added in PBS/2% Tween-20 for 30 min prior to final washes and mounting (Input protocol)

Cells were fixed in 4% paraformaldehyde for 15 minutes on ice, washed in PBS, permeabilized with 0.1% Triton X-100 in PBS for 45 minutes, and incubated for 30 minutes with TRITC-labeled phalloidin diluted 1:1000 in PBS. DNA was stained with DAPI (1 μg/ml in PBS) for 15 minutes (Protocol 1)

Cells were labeled with Alexa Fluor 546 Phalloidin (1:50; Molecular Probes, Eugene, OR) for 1 hour and then washed in phosphate buffer saline (PBS; 3 times for 5 minutes). TOTO-3 iodide (1:200; Molecular Probes, Eugene, OR) was then added to each construct to stain the cell nuclei (Protocol 2)

The samples were first permeabilized with 0.1% Triton-X for 20 min then washed with PBS and incubated with AlexaFluor-488 Phalloidin (Cat# A12379, Life Technologies), and TOPRO-3 (Cat# T3605, Life Technologies) or DAPI stains for 1 hr to label F-actin and nuclei, respectively (Protocol 3)

For the F-actin staining, cells were fixed with 2% formaldehyde for 15 min at room temperature and permeabilized with PBS buffer containing 0.1% Triton- X100. The cells were further incubated with Phalloidin CruzFluor 488 Conjugate (Santa Cruz Biotechnology) and mounted in Vectashield mounting medium with DAPI (Protocol 4)

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