Adult male Sprague Dawley rats (n=6, 200–250 g, 6–7 weeks old; Shanghai SLAC Laboratory Animal Co., Ltd., Shanghai, China) were housed under a 12-h light/dark cycle, at 23–25°C and 45–50% humidity and provided with free access to food and water. All surgical and experimental procedures were approved by the Animal Ethics Committee of Fudan University (Shanghai, China).

To prepare a 1% sodium pentobarbital solution, 5 g sodium pentobarbital (cat. no. 69020181; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was dissolved in 500 ml distilled (d)H₂O, and the solution was stored at 4°C in the dark. To prepare 1 l of 4% paraformaldehyde, 40 g paraformaldehyde was added to 1 l of 1X phosphate-buffered saline (PBS) and heated gradually to 60°C with continuous stirring to dissolve the paraformaldehyde. The pH was subsequently adjusted to 7.4 with NaOH. To prepare a 10 or 30% sucrose solution, 10 or 30 g sucrose (cat. no. 10021418; Sinopharm Chemical Reagent Co., Ltd.) was added to 100 ml dH₂O. To prepare 1 l of antigen unmasking buffer (10 mM sodium citrate), 2.94 g sodium citrate tribasic salt dihydrate (C₆H₅Na₃O₇·2H₂O, cat. no. 10019418; Sinopharm Chemical Reagent Co., Ltd.) was added to 1 l dH₂O. The pH was adjusted to 6.0 and the solution was subsequently filtered (pore diameter, 75 µm). To prepare 1 l of 20X saline-sodium citrate (SSC), 175.2 g NaCl and 88.2 g sodium citrate tribasic salt dihydrate were dissolved in 800 ml dH₂O. The pH was adjusted to 7.0, and dH₂O was added to bring the volume to 1 l. To prepare 100 ml of prehybridization buffer, 3 g bovine serum albumin (BSA; cat. no. 69003433; Sinopharm Chemical Reagent Co., Ltd.) was added to 100 ml of 4X SSC. The resulting solution was used to prepare 0.1, 1, 2 and 4X SSC. To prepare 10 ml of hybridization buffer, 1 g dextran sulfate (cat. no. S14047; Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China) and 1 ml deionized formamide (cat. no. 30091218; Sinopharm Chemical Reagent Co., Ltd.) were added to 9 ml of 4X SSC. To prepare 100 ml of blocking buffer, 1 g BSA and 0.1 ml Tween-20 were added to 100 ml of 1X PBS. To prepare 50 ml of 4′,6-diamidino-2-phenylindole (DAPI) staining solution, 0.5 µl DAPI stock solution was diluted in 50 ml of 1X PBS.

All DNA oligonucleotides, including three probes for rat lncRNA malat1 (Malat1-a, -b and -c) and one probe for GAPDH (positive control; Table I), were designed and synthesized and labeled on the 5′-end with Alexa Fluor 633 by Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Probes were stored at −20°C in powder form. To prepare a 250 nM working solution, probes were diluted in hybridization buffer. Antibodies against NeuN, Iba1 and GFAP were used as biomarkers of neurons, microglia and astrocytes, respectively (11). The primary and Alexa Fluor 488-conjugated secondary antibodies used for immunofluorescence were purchased from Abcam (Cambridge, MA, USA; Table II) and diluted with blocking buffer to 1:500 and 1:400, respectively.

Rats were anesthetized with an intraperitoneal injection of 1% sodium pentobarbital solution (40 mg/kg) and transcardially perfused with 1X PBS and 500 ml of 4% paraformaldehyde in phosphate buffer (0.1 M, pH 7.4). The spinal cord was harvested at the cervical level and specimens were immersed in 4% paraformaldehyde for 4 h at 4°C. The tissues were then sequentially immersed in 10 and 30% sucrose until the spinal cords fell to the bottom. Spinal cords were embedded in optimal cutting temperature compound (cat. no. 4583; Sakura Finetek USA, Inc., Torrance, CA, USA) and mounted on a microtome stage. Horizontal sections (10 µm) were mounted onto pre-warmed poly-l-lysine-coated slides (cat. no. P4981; Thermo Fisher Scientific, Inc.).

Following desiccation for 20 min at room temperature, slides were rinsed twice with 1X SSC (5 min/wash). The slides were placed into a Coplin jar filled with antigen unmasking buffer, which was placed into an autoclave and heated to 100°C for 5 min and subsequently cooled to room temperature. Sections were then washed three times with 1X SSC (5 min/wash). The tissue sections were dehydrated in a graded series of ethanol (50, 70, 90 and 100%; 3 min each) and air-dried for 10 min.

For comparison, some slides were treated with PK instead of treatment with antigen unmasking buffer and autoclaving, according to the method described by Nehmé et al (9). For this method, the slides were immersed in a buffer containing 0.1 M Tris-HCl (pH 8), 50 mM EDTA (pH 8) and 10 µg/ml PK (Roche Diagnostics, Basel, Switzerland) for 25 min at 37°C.

The prehybridization buffer was warmed to ~47°C [usually 22–25°C below the melting temperature (T_m) of probes] and 200 µl warmed prehybridization buffer was pipetted onto each slide and prehybridized for 20 min in a humid chamber (47°C). The probes (Malat1-a, -b and -c and GAPDH) were diluted with hybridization buffer and denatured at 65°C for 10 min, then stored on ice.

The prehybridization buffer was removed and 100 µl aliquots of each probe (or same volume of hybridization buffer without probes as negative control) were added to separate slides and hybridized overnight (4–16 h) at the same temperature used in the prehybridization step. From this step on, all slides were kept in the dark.

Following hybridization, the slides were rinsed with the following solutions: 4X SSC (twice at 5 min/wash), 2X SSC (5 min), 1X SSC (5 min), and 0.1X SSC (5 min) at the same temperature as in the hybridization step. The slides were then rinsed with 1X PBS for 5 min at room temperature.

Following in situ hybridization, the sections were blocked in blocking buffer for 30 min at 37°C. From this step on, sections were kept in a humid chamber to protect the tissue sections from drying out. The blocking buffer was removed and 200 µl primary antibody diluted in blocking buffer was added (Table II), and the sections were incubated overnight at 4°C in a humid chamber. Sections were rinsed three times with 1X PBS (5 min/wash), then incubated with corresponding secondary antibody (200 µl diluted in blocking buffer) for 2 h at 37°C. Slides were subsequently rinsed three times with 1X PBS (5 min/wash). At this point, all FISH and immunofluorescence staining steps were complete. However, if the nucleus needed to be observed, nuclear staining with DAPI was performed. Finally, a few drops of antifade (cat. no. P36965; Thermo Fisher Scientific, Inc.) were added to the slides and a coverslip was placed. The slides were sealed with nail polish.

Images were captured with a confocal laser-scanning microscope (FluoView™ FV1000; Olympus Corporation, Tokyo, Japan) equipped with a digital camera and image analysis system (FV10-ASW 4.0; Olympus Corporation). All images were analyzed using Image J 1.44 (National Institutes of Health, Bethesda, MD, USA). Brightness and contrast were adjusted. Positive cells and the background surrounding positive cells were selected as regions of interest. The ratio of integral optical density (IOD) between the positive cells and background signal was calculated using Image J 1.44. All statistical analyses using Student's t-test and histograms were completed with GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

Satisfactory fluorescent signals were obtained from all three malat1 probes (Malat1-a, -b and -c; Fig. 1A-C). During FISH, GAPDH served as a positive control and sections without probes (hybridization buffer only) served as negative controls (Fig. 1D and E). The IOD ratio of Malat1-a, -b, -c group was respectively compared with negative control using Student's t-test. All three Malat1 probes were demonstrated to be effective (n=6, P<0.05); however, the Malat1-b probe exhibited the highest signal amplitude (Fig. 1F). Therefore, Malat1-b was used for subsequent experiments.

Two antigen retrieval methods, autoclaving and PK treatment, were compared. Using the same imaging parameters, both methods achieved notable FISH signals (Fig. 2). The IOD ratio of positive cells to background signal was calculated for each group, and did not differ significantly between the two groups (Fig. 2C).

The Malat1-b probe was used to detect the expression of malat1 (Fig. 3; labeled red). Using immuno-FISH, malat1 and three different proteins were successfully co-detected. NeuN-positive cells were double labeled with malat1 (green-red merge; Fig. 3A), while cells positive for glial fibrillary acidic protein and ionized calcium binding adaptor molecule 1 (Iba-1) were a single color (green; Fig. 3B and C). These results indicated that Malat1 is expressed in neurons but not in microglia or astrocytes.