Vectamount aq

Transwell inserts (Corning) with 8 μm pores were coated with fibronectin (20 μg/mL, Roche, Germany) for 30 min at 37°C. 700 μL of conditioned media was added to each well of a 24 well plate (Corning) followed by addition of fibronectin coated inserts. To each insert 1x10⁵ THP-1 monocytes were added and cells were allowed to migrate for 12 hours in 5% CO₂ at 37°C. TNF-α (10 μg/mL) was used as control and IgG (1 μg/mL) was used as isotype control. For staining inserts were washed with phosphate buffered saline (Sigma-Aldrich) followed by fixation with 3.7% formalin solution (Roth) for 10 min. Following fixation inserts were stained with 0.2% crystal violet (Sigma-Aldrich). Cells on the top of the insert were removed using a cotton swab and only cells which migrated across the membrane were analyzed. The membrane of the transwell insert were carefully cut using a scalpel and mounted on a glass slide using mounting medium (VectaMount™ AQ, Vector Laboratories). 10 high power field (HPF) digital images were taken using DMI 3000B microscope with a DFC300FX camera (Leica Microsystems). Cells per HPF were counted using ImageJ software.

Whole testes from mice were fixed in Bouin’s fluid for 8 hours. Tissues were dehydrated in a graded series of ethanol and embedded in paraffin. Sections that were 7 μm thick were processed for stainings. Sections were deparaffinized and rehydrated before antigen retrieval, followed by processing for antibody staining against the Leydig cell marker protein INSL3 (orb18041, AA range: 10–50, 1:100, Biorbyt) or the macrophage marker protein F4/80 (MCA497GA, Cl:A3-1, 1:500, AbD). The stainings were developed using alkaline phosphatase (Dako, 1:200) followed by counterstain with Harris’ hematoxylin and mounting under VectaMount AQ (Vector Laboratories). For immunofluorescence on tissues, sections were immunostained with antibodies against STAR (8849, 1:1000, clone D10H12, Cell Signaling Technology) or CCL2 (orb36895, 1:1000, Biorbyt) antibodies followed by detection with secondary antibodies (anti–rabbit IgG, 711295152, 1:400, Jackson ImmunoResearch; or anti–mouse IgG, SAB3701033, 1:400, MilliporeSigma) and counterstained with DAPI. For immunofluorescence on cells, cell smears were fixed in 4% paraformaldehyde and permeabilized in 70% ethanol. After that, slides were immunostained with antibodies against STAR, CCL2, or VDAC-1 (SAB5201374, clone S152B-23, 1:200, MilliporeSigma) followed by conjugation with the abovementioned secondary antibodies and counterstain with DAPI.

Brains from perfused mice were frozen in OCT (Fisher Scientific) and ten-micron thick sections were processed for immunohistochemistry. Briefly, sections were fixed in ice cold 95% ethanol for 15 min and washed in PBS several times. This was followed by washes in TBS with 0.1% Tween (TBST) and incubation for 10 min with 3% H₂O₂ to block endogenous peroxidase. After washing, blocking buffer was added for 1 h (10% normal goat serum in PBS). Primary antibody was added overnight at 4 °C: purified rat anti-mouse CD4, anti-mouse CD8 and anti-mouse F4/80 (all from eBiosciences), diluted 1:100 in PBS 2% normal goat serum. After washes in TBST, the biotinylated secondary antibody (anti-rat IgG, mouse absorbed, Vector) was added for 1 h, diluted 1:200 in PBS 2% normal goat serum. After washes in TBST, the Vectastain ABC reagent was used (Vector) following manufacturer’s instruction. Then, DAB (Sigma) was added as a substrate and, after incubation for 8 min in the dark and several washes in distilled water, sections were counterstained with Harris hematoxylin for 20 seconds, in lithium carbonate for 30 sec, washed in several changes of distilled water and mount with VectaMount AQ (Vector).

Lungs were harvested at 2 dpi and placed in PBS-buffered formalin, blocked in paraffin, 10-μm tissue sections were cut, and placed on glass slides. Lung sections where deparaffinized in Xylenes (3X 10’), and washed in 100% Ethanol (2X 5’). After progressive rehydration (95% Ethanol 2X 5’, 70% Ethanol 1X 5’), slides were rinsed 3 times in 1X PBS. Antigen unmasking was performed using a reagent from Vector (H-3300, 2.5ml in 250ml H2O), and microwaving 2X for 5 minutes, followed by a 20 minute cool down, and 2 PBS1X rinses at RT. Lungs sections were outlined using an Elite Mini PAP Pen (Diagnostic BioSystems K 042), and incubated in blocking buffer (1XPBS with 1% BSA) for 30’. Incubation with primary antibodies primary antibodies was performed overnight at 4°C in a humid chamber followed by 3X PBS rinses. Secondary incubation was performed in blocking buffer followed by 3X rinses in PBS. Slides were mounted using VectaMount AQ (Vector, H 5501).

To quantify LGR5+ cells in jejunal tissues, formalin-fixed tissue sections were stained with anti- LGR5 antibodies using the Mach3 Rabbit AP-Polymer Detection Kit (Biocare Medical, USA) as performed earlier (31 (link)). The paraffin-embedded tissue sections were deparaffinized and epitope retrieved by heating the tissue sections in low pH citrate buffer (Vector Laboratories, USA) using a microwave. After blocking with a serum-free protein blocker (Agilent Dako, USA) for 30 min, the tissue sections were incubated with anti-LGR5 primary antibodies (Supplementary Table 1). The negative control slide consisted of rabbit Ig fractions (R&D Systems, USA) used at the same isotype and concentration as LGR5 antibodies to determine the background intensity and staining. The tissue sections were subsequently treated with the kit’s probe and polymer and finally developed using BCIP/NBT (Agilent Dako) chromogen system. The slides were then mounted with Vecta Mount AQ (Vector Laboratories). An average of 19–20 view fields (0.116mm²/view field under a 400 x magnification) were enumerated in each of the slides to quantify LGR5+ cells manually. The tissue sites for this evaluation were selected randomly. Tissues were analyzed by two different blinded individuals to avoid bias and averaged prior to the statistical analysis.