Entellan new

To visualize degenerative neurons, brain sections were subjected to FJC staining according to kit instructions (Millipore, USA). Briefly, the slides were fixed with 4 % paraformaldehyde for 10 min then immersed in 1 % NaOH in 80 % ethanol for 5 min. After rinsing for 2 min each in 70 % ethanol and ddH₂O, slides were incubated in 0.06 % potassium permanganate solution for 10 min, rinsed in ddH₂O, then transferred to the staining solution for 10 min. Post washing, slides were air-dried at 50 °C for 30 min then cleared in xylene and mounted with Entellan New (Sigma-Aldrich, USA).

Histological analyses were carried out using Technovit 7100 (Kulzer Technik, Germany) according to the protocol of Yeung and Chan (2015) (link) with slight modifications. Briefly, ovaries collected at 0, 2, 4, and 10 DAA were fixed as described in Section 2.4 and then dehydrated by passing them sequentially through graded ethanol (70%/2 h → 80%/2 h → 90%/2 h → 100%/overnight). This was followed by three infiltration steps in ethanol:Technovit solutions at different concentrations (2:1/2 h → 1:1/2 h → 1:2/2 h), and overnight immersion in pure Technovit. The ovaries were then embedded in a mixture of Technovit and Hardener II (15:1 v/v) and allowed to polymerize at room temperature for 12 h before sectioning at a thickness of 5 μm using a rotary microtome machine (Leica RM2235, Leica Biosystem Ltd., China). Finally, the sections were mounted in water on a glass slide, dried at 40°C, and stained with toluidine blue (Sigma-Aldrich T3260, Merck, USA). A drop of Entellan New (Sigma-Aldrich 107961, Merck, USA) was added to the slide before observation under a light microscope (Olympus BX50). At least three ovaries were observed for each style removal treatment. To estimate fruit growth, cell layer and cell volume measurements were conducted on the microscope images using ImageJ software.

Immunohistochemical analysis was carried out using the anti-mouse IL-1β rabbit polyclonal antibody (ab205924) (Abcam; Cambridge, UK). An anti-TNF-α rabbit polyclonal antibody (26405-1-AP) and an anti-IL-6 rabbit polyclonal antibody (21865-1-AP) were purchased from Proteintech (Rosemont, IL, USA). Sections (3–5 μm thick) were cut from formalin-fixed paraffin-embedded tissues, deparaffinized in xylene and rehydrated through a decreasing concentration of ethanol solutions. Endogenous peroxidase activity was blocked by addition of 0.3% H₂O₂ in methanol for 30 min. Before immunostaining, antigen retrieval was carried out by heating tissue sections in a microwave in 10 mmol/L Tris-HCl buffer (pH 8.0) containing 1 mmol/L ethylenediaminetetraacetic acid (EDTA). Sections were blocked with 1% normal bovine serum in 50 mmol/L Tris-buffered saline (TBS) (pH 7.6) and then incubated with primary antibodies diluted in blocking buffer. Binding was detected using EnVision+ Rabbit/HRP (Dako, Carpinteria, CA, USA), and positive signals were revealed by the addition of diaminobenzidine tetrahydrochloride. Tissue sections were counterstained with hematoxylin and then mounted in Entellan new (Sigma-Aldrich, St. Louis, MO, USA).

The frozen sections were incubated at room temperature, washed with water for 2 to 3 min, and stained with hematoxylin (Muto Pure Chemicals Co., Ltd., Tokyo, Japan) for 8 min. After washing with water for 15 min, the sections were stained with eosin for 1 min. After washing with running water, the sections were dehydrated stepwise using various concentrations of ethanol and then incubated in xylene three times. The sections were sealed with Entellan New (Sigma, #107961), observed, and photographed under an optical microscope (OLYMPUS BX50 [Evident, Tokyo, Japan]).

Following perfusion, spinal cord, brain and brainstem were removed, postfixed for 24 h, cryoprotected in 30% sucrose for 48–72 h, frozen, and cut in coronal sections of 40 μm at −20°C in a cryostat. Free floating sections were incubated with c-Fos rabbit polyclonal antibody (1:2000; Santa Cruz Biotechnology, catalog #sc-52) for 48 h at 4°C. Later, they were rinsed and incubated with a biotinylated donkey-anti rabbit antibody (1:500, Jackson ImmunoResearch, catalog #711-065-152, RRID:AB_2340593) and avidin–biotin complex (1:500, Vector Laboratories, catalog #PK-4000), for 2 h each at room temperature. Product visualization was obtained with 0.01% diaminobenzidine, 0.05% nickel ammonium sulfate and 0.01% hydrogen peroxide for 15 min. All sections were mounted, dehydrated, and cover-slipped with microscopy Entellan New (Merck/Sigma-Aldrich, catalog #107961).

As previously described (34 (link)) for H&E (Haematoxylin and Eosin) staining, we embedded lung lobes in Tissue-Tek O.C.T. compound (Sakura Fintek, USA) and then froze them in liquid nitrogen. The molds were sectioned into 5 μm slices followed by staining with filtered 0.1% Mayers Hematoxylin (Sigma; MHS-16) and rinsing in cool running ddH₂O. Next, they were immersed in 0.1% HCl water and then in undiluted Eosin. Subsequently the slides were rinsed in cool running ddH₂O and dehydrated in 70% EtOH and then in 95% EtOH. Finally, they were placed in xylene and this was repeated in fresh xylene mount and coversliped with Entellan new (Sigma-Aldrich, Germany).