. 2022 May 5;133(1):41–59. doi: 10.1152/japplphysiol.00088.2022

Table 5.

Example protocols for hematoxylin/eosin and laminin staining on electroporated skeletal muscles

Hematoxylin and Eosin	Laminin
If slides were in −80°C, allow to equilibrate to room temperature. Incubate the slides with hematoxylin solution in a staining jar for 10 min to stain the nuclei. Transfer the slides to a staining jar with running water (tap water is fine) till the water is clear. Transfer the slides to a staining jar with eosin solution for 3 min. Successfully transfer the slides into staining jars with 70% ethanol for 20 s, 90% ethanol for 20 s, 100% ethanol for 1 min and citrisolv for 3 min. Take out slides from citrisolv and place the slides in the fume hood until the slides are dry. Mount the slides with permount media and coverslip. Store the slides at room temperature overnight.	If slides were in −80°C, allow to equilibrate to room temperature. Draw a box/circle around sections on the slide using a Pap Pen. Incubate slides in 1× PBS + 1% Triton for 10 min at room temperature (RT) to permeabilize. Rinse with 1× PBS, two times, 5 min each. Block with 5% normal goat serum in PBS for 15 min at RT. Rinse once with 1× PBS for 5 min. Make up enough laminin antibody to cover slides. Laminin is added at 1:500 in 5% normal goat serum. Incubate with laminin antibody for 2 h at RT. Rinse with 1× PBS, two times, 5 min each. Make up enough secondary antibody to cover slides. Secondary is added at 1:333 in 5% normal goat serum. We commonly use Alexa Fluor 555 goat anti-rabbit. Incubate with secondary antibody for 1 h at RT. After 1 h, remove secondary antibody and coverslip the microscope slides using appropriate mounting medium.

Hematoxylin and Eosin

Laminin

If slides were in −80°C, allow to equilibrate to room temperature.
Incubate the slides with hematoxylin solution in a staining jar for 10 min to stain the nuclei.
Transfer the slides to a staining jar with running water (tap water is fine) till the water is clear.
Transfer the slides to a staining jar with eosin solution for 3 min.
Successfully transfer the slides into staining jars with 70% ethanol for 20 s, 90% ethanol for 20 s, 100% ethanol for 1 min and citrisolv for 3 min.
Take out slides from citrisolv and place the slides in the fume hood until the slides are dry.
Mount the slides with permount media and coverslip. Store the slides at room temperature overnight.

If slides were in −80°C, allow to equilibrate to room temperature.
Draw a box/circle around sections on the slide using a Pap Pen.
Incubate slides in 1× PBS + 1% Triton for 10 min at room temperature (RT) to permeabilize. Rinse with 1× PBS, two times, 5 min each.
Block with 5% normal goat serum in PBS for 15 min at RT. Rinse once with 1× PBS for 5 min.
Make up enough laminin antibody to cover slides. Laminin is added at 1:500 in 5% normal goat serum. Incubate with laminin antibody for 2 h at RT. Rinse with 1× PBS, two times, 5 min each.
Make up enough secondary antibody to cover slides. Secondary is added at 1:333 in 5% normal goat serum. We commonly use Alexa Fluor 555 goat anti-rabbit. Incubate with secondary antibody for 1 h at RT.
After 1 h, remove secondary antibody and coverslip the microscope slides using appropriate mounting medium.