Ab32423

Paraffin-embedded NSCLC tissues were dissected into consecutive slices and analyzed using IHC assay as previously described [33 (link)] with antibodies against FABP7 (Abcam, ab32423, Cambridge, MA, USA 1:50), MMP7 (Abcam, ab4044, Cambridge, MA, USA 1:40), and MYC (HuaBio, 0912-2, Hangzhou, China, 1:200), respectively. The immunostaining degree of indicated proteins was evaluated and scored by two independent observers with both the proportions of positively stained tumor cells and the staining intensities. The proportions of positively stained tumor cells were scored as: 0 (no positive tumor cells), 1 (<5%), 2 (5–25%), 3 (25–50%), and 4 (>50%). The intensities of staining were determined as: 0 (no staining), 1 (weak staining = light yellow), 2 (moderate staining = yellow-brown), and 3 (strong staining = brown). The staining index (SI) was calculated as the function of staining intensity × percentage of positive tumor cells, resulting in scores of 0, 1, 2, 3, 4, 6, 8, 9, and 12. Cutoff values for high and low expression of target proteins were chosen based on a measurement of heterogeneity using the log-rank test with respect to overall survival. SI ≥ 4 indicates high expression and SI ≤ 3, low expression. Chi-squared (χ2) tests were used for contingency tables.

Frozen brain sections were dried at room temperature, and then pretreated with 0.3% H₂O₂ for 15 min to deactivate endogenous peroxidase. Sections were blocked with 3% normal sheep serum with 0.1% Tween 20 at room temperature for 2 h. Sections were then incubated in primary antibodies [rabbit anti-NeuN (1:500; Abcam, ab177487), rabbit anti-SOX2 (1:500; Millipore, ab5603), rabbit anti-TBR2 (1:500; Abcam, ab23345), mouse anti-PROX1 (1:200; Millipore, MAB5654), and rabbit anti-BLBP (1:500; Abcam, ab32423)] in blocking buffer overnight at 4 °C, followed by addition of the avidin-biotin-peroxidase complex (1:50; VECTASTAIN Elite ABC system, Vector Laboratories). Peroxidase was reacted in 3,3′-diaminobenzidine (5 mg/ml) and 0.075% H₂O₂ in Tris-HCl (pH 7.2). Sections were dehydrated in gradient ethanol (75% ethanol, 95% ethanol, 100% ethanol, and 100% ethanol, each for 5 min), and cleared twice in xylene for 5 min, then mounted in the neutral balsam.

For immunofluorescence, every 6th section was collected and washed in PBS. The sections were then incubated for 2 h in blocking buffer PBS-plus (10% donkey serum, 0.2% Triton-X 100 in PBS) to block unspecific binding sites and to permeabilize the tissues. After the blocking step, the sections were incubated overnight at 4°C with the primary antibodies diluted in incubation buffer (PBS with 3% donkey serum). The following primary antibodies and concentrations were used: rabbit-anti GFAP (Z0334, 1:1000, Dako), goat-anti Doublecortin (sc-8066, 1:250, Santa Cruz Biotechnology), mouse-anti Prox1 (MAB5654, 1:500, Chemicon International), rabbit-anti Ki67 (NCL-Ki67p 1:500, Novocastra), rabbit-anti Tbr2 (23345, 1:800, Abcam), goat-anti Sox2 (sc-17320, 1:200, Santa Cruz Biotechnology), goat-anti NeuroD (sc-1086, 1:400, Santa Cruz), rabbit-anti BLBP (ab32423, 1:400, Abcam). After several washes in PBS, the sections were incubated for 4 h at room temperature in secondary antibodies diluted in incubation buffer. The concentration of secondary antibodies CY3 (711-495-152, Jackson Immuno Research) and CY5 (715-175-15, Jackson Immuno Research) was 1:500. After the incubation, sections were washed several times in PBS and incubated in DAPI (861405, 1:4000, Invitrogen) for 10 min, then washed again in PBS and mounted on slides in Aqua Poly/Mount.

It was performed as previously described (Wu et al., 2015 (link); Yang et al., 2019 (link)). Briefly, frozen sections were washed 10 min with 0.5% phosphate-buffered saline with Tween 20 (PBS-T) for three times and then blocked with 3% bovine serum albumin for 1 hr. After that, sections were incubated overnight at 4°C with the primary antibodies as follows: Calbindin (C9848, Sigma, 1:400), NeuN (MAB377, Millipore, 1:400), brain lipid binding protein (BLBP) (ab32423, Abcam, 1:500), Rack1 (R1905, Sigma, 1:400). The sections were washed 10 min with 0.5% PBS-T for three times again and subsequently subjected to Alexa Fluor-conjugated secondary antibodies (Biotium, 1:500). Nuclear staining was visualized with a mounting medium with 4′,6-diamidino-2-phenylindole (ZSGB-BIO). All images were taken from a laser scanning confocal microscope (Olympus FV1200) and then were processed and analyzed by FV10-ASW or Image Pro Plus 6.0 software.