Immunofluorescence
Two mammary tissue blocks (0.3 cm3) were immediately
sampled after the mice were sacrificed and then stored in liquid
nitrogen for tissue immunofluorescence. Briefly, 5-µm cryosections of
mammary glands were fixed with 4% paraformaldehyde in PBS for 40 min at
room temperature, followed by incubation with PBS containing 5% BSA
(ThermoFisher Scientific, Waltham, MA, USA) to block nonspecific
interactions and then treatment with primary antibodies diluted by BSA
overnight at 4 ℃. After washing with PBS, the sections were exposed to
secondary antibodies and diluted by 5% BSA for 1 h at room temperature.
Controls were treated in the same manner, except that primary antibodies
were not applied. After washing with PBS and staining with DAPI for 5
min, images of stained sections were obtained using a fluorescence
microscope.
Transmission
electron microscope analysis
Two mammary gland tissue blocks (10 mm3) collected
from each mouse were immediately stored in centrifuge tubes and filled
with electron microscope fixative for
transmission
electron microscopy (TEM) (Gang et al., 2019). Briefly, cell samples
were harvested and filled with an electron microscope fixative for TEM.
Both tissue and cell samples were dehydrated with ethanol (30, 50, 70,
80, 90, and 100% ethanol) with 15 min per step at room temperature.
Cells were then embedded in epoxy resin acetone mixtures (2:1) for 2 h
and in pure resin at 37 °C overnight. Next, ultra-thin sections were cut
using an ultramicrotome (Leica EM, Germany), stained with 1% uranyl
acetate and lead citrate, and examined with TEM (Hitachi H-7650, Japan).