Immunostaining of Cell Types in Tissue Samples

Immunofluorescent staining was performed following previously described procedure26 (link) with the following primary antibodies: anti-CD68 (1:100; Abcam), anti-alpha smooth muscle actin (1:200; Abcam), anti-Collagen I (1:200; Abcam). The following secondary antibodies were used for double staining: Goat polyclonal secondary antibody to rabbit IgG (FITC; Abcam) and donkey anti-goat IgG (Cy5 ^®; Abcam). Nuclei were stained with DAPI (Sigma-Aldrich, USA). The specimens were pretreated through heating followed by blocking with 1% BSA (Sigma-Aldrich) and then incubated with the primary antibody at 4°C overnight. After primary antibody staining, the specimens were washed with PBS, incubated with a secondary antibody, stained with DAPI and mounted on coverslips. Serial sections of the specimens were observed with a digital pathology scanner (APERIO VERSA 8; Leica).

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Wang Y., Zhang H., Zhou M., Yi X., Duan P., Yu A, & Qi B. (2020). Autologous Fat Grafting Promotes Macrophage Infiltration to Increase Secretion of Growth Factors and Revascularization, Thereby Treating Diabetic Rat Skin Defect. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy, 13, 4897-4908.

Publication 2020

anti-CD68 anti-alpha smooth muscle actin anti-Collagen I FITC DAPI BSA APERIO VERSA 8

Alpha actin Anti igg Antibodies Antibody Collagen 1 Dapi Donkey Fitc Goat Igg antibody Immunofluorescent Nuclei Rabbit Scanner Smooth muscle

Corresponding Organization : Wuhan University

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Variable analysis

independent variables

Primary antibodies: anti-CD68 (1:100; Abcam), anti-alpha smooth muscle actin (1:200; Abcam), anti-Collagen I (1:200; Abcam)

dependent variables

Immunofluorescent staining pattern of CD68, alpha smooth muscle actin, and Collagen I

control variables

Heating pretreatment of specimens
Blocking with 1% BSA (Sigma-Aldrich)
Incubation with primary antibodies at 4°C overnight
Washing with PBS after primary antibody staining
Incubation with secondary antibodies
Staining with DAPI
Mounting on coverslips
Observation with a digital pathology scanner (APERIO VERSA 8; Leica)

controls

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

Annotations

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