Table 2 Lamin b1 immunofluorescence staining protocol for endometriosis tissue.

Day 1

1

Cryostat 4-µm thick sections previously sliced on charged slides and stored at − 80 °C should be collected and let dry at room temperature (23–26 °C)

2

Draw a circle around each tissue section using hydrophobic barrier pen to keep reagents localized on tissue specimens—avoiding waste of materials and preventing mix of reagents

3

Cover the tissues sections with a 4% paraformaldehyde solution and let them incubate for 15 m for tissue fixation

4

Wash sections three times with 0.5% Tween solution—no need for time incubation at this wash step

5

Incubate sections with a 0.2% Triton-X 100 solution for 15 min

6

Wash sections three times with 0.5% Tween solution—no need for time incubation at this wash step

7

Block the tissue covering the slides with a 5% BSA solution for 30 min

8

Wash sections three times with 0.5% Tween solution—no need for time incubation at this wash step

9

Prepare the primary antibodies solution following the dilution displayed below:

Dilution of lamin b1 (1:50) in prediluted E-cadherin

2 µL of lamin b1 in 100 µL of prediluted E-cadherin

Note: If the second biomarker chosen for colocalized immunofluorescence is not prediluted, a 0.5% Tween and 5% BSA solution should be used to meet the required dilution for both primary antibodies

10

Cover the tissue sections of the positive control and the interest tissue with the primary antibody solution, while the negative control should be covered with PBS

11

Gently put the slides in a humidified chamber to overnight incubation of 24 h in 4 °C

Day 2

1

Take the chamber containing the slides from the freezer and gently place it at room temperature (23-26 °C)

2

Wash sections for three times with 0.5% Tween solution for 5 min—this step is particularly important to avoid overlabeling by the primary antibodies

3

Prepare the secondary antibodies solution together with 0.5% Tween and 5% BSA solution following the dilution displayed below:

Dilution secondary antibodies (Alexa Fluor^® 488 and Alexa Fluor^® 647)—1:400

1 µL of Alexa Fluor® 488 + 1 µL Alexa Fluor® 647 in 400 µL of 0.5% Tween and 5% BSA solution

4

Cover all the tissue sections with the secondary antibody solution for 1 h at room temperature. If available, chose a dark chamber to rest the slides during this incubation period to avoid photobleaching

5

Wash sections for three times with 0.5% Tween solution for 5 min—this step is particularly important to avoid overlabeling by the secondary antibodies

6

Let the slides dry at room temperature

7

Stain nuclei with DAPI and mount with mounting medium