All the fragments were ligated into pcDNA3.1(+) plasmid, and the constructed plasmids were then verified by DNA sequencing. The pCre-luc (Santa Cruz, CA, USA) was kindly gifted by Xin Xie’s Lab of Tongji University. Human α-melanocyte stimulating hormone (α-MSH) and human adrenocorticotropin ACTH (1–24) was synthesized by Genescript (Nanjing, China). We purchased the paraformaldehyde (4%), phosphate buffered saline (PBS), bovine serum albumin, non-fat milk powder and β-mercaptoethanol from Sangon Biotech (Shanghai, China). TRNzol Universal Reagent and FastQuant RT Kit (with gDNase) were obtained from Tiangen Biotech (Beijing, China). Tetramethylbenzidine (TMB) chromogen solution was purchased from Beyotime® Biotechnology (Shanghai, China). Hydrochloric acid and sulfuric acid were obtained from Sinopharm Chemical Reagent Co., Ltd (Beijing, China). The antibodies used in this study included Rabbit anti-Flag (Cell Signaling Technology, USA), Mouse anti-HA (Sigma Aldrich, MO, USA), Mouse anti-Flag (Abcam, Cambridge, UK), Goat anti-Mouse IgG (horseradish peroxidase (HRP)-conjugated) (ABclonal Biotech Co., Ltd, Wuhan, China), and Goat Anti-Rabbit Alexa-Fluor 594 (Abcam). All primers used for full-length gene amplification, reverse transcription PCR (RT-PCR), and real-time quantitative PCR are listed in Supplemental Table 1
Goat anti rabbit alexa fluor 594
Manufactured by Abcam
Sourced in United Kingdom
Goat anti-rabbit Alexa Fluor 594 is a secondary antibody conjugated with the Alexa Fluor 594 fluorescent dye. It is designed to detect and visualize rabbit primary antibodies in various immunoassays and microscopy applications.
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Lab products found in correlation
Goat anti mouse alexa fluor 488, by Abcam (6 mentions)
Alexa fluor 488 goat anti rabbit, by Abcam (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Fluorescence microscope, by Leica (2 mentions)
Goat anti mouse alexa fluor 594, by Abcam (2 mentions)
Mouse anti ha, by Merck Group (1 mentions)
Sulfuric acid, by Sinopharm (1 mentions)
Hydrochloric acid (hcl), by Sinopharm (1 mentions)
Pcre luc, by Santa Cruz Biotechnology (1 mentions)
Non fat milk powder, by Sangon (1 mentions)
Mouse anti flag, by Abcam (1 mentions)
Fastquant rt kit with gdnase, by Tiangen Biotech (1 mentions)
Rabbit anti flag, by Cell Signaling Technology (1 mentions)
Trnzol universal reagent, by Tiangen Biotech (1 mentions)
Goat anti rabbit igg, by Southern Biotech (1 mentions)
Alexa fluor 647 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Ab15537, by Abcam (1 mentions)
Ab59479, by Abcam (1 mentions)
Tcs sp8 confocal microscope, by Leica (1 mentions)
Phalloidin ifluor 488, by Abcam (1 mentions)
14 protocols using goat anti rabbit alexa fluor 594
1
Plasmid Construction and Validation
2
Generation and Characterization of GPER Antibodies
3
Multimarker Immunofluorescence Visualization
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Cells were fixed, permeabilized and incubated with the following primary antibodies: mouse monoclonal to CD9, sc-59,140; mouse monoclonal to CD81, sc-166,029; mouse monoclonal to CD63, ab59479, rabbit polyclonal to BMP11 (=GDF11), ab220951, rabbit polyclonal to TGF-β3, ab15537, from Abcam; and rabbit polyclonal to Zeb1, HPA027524, from Sigma-Aldrich. Phalloidin-iFluor 488 (Abcam) was used to counterstain the cell’s actin cytoskeleton. The secondary antibodies used were: goat anti-rabbit Alexa Fluor 594 (Abcam) and goat anti-mouse Alexa Fluor 647 (Invitrogen). Nuclei were stained with DAPI. Cells were visualized in a TCS SP8 confocal microscope (Leica).
4
Quantifying Neutrophil CFTR and LAMP1
5
Immunostaining of M1 and M2 Macrophages
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BMMs were seeded onto confocal culture dishes (1 × 105 cells/well) and incubated with vehicle and 5 or 10 μM DAC. After culturing for 3 days, 4% paraformaldehyde in PBS was used to fix the cells for 15 min at room temperature. The cells were then washed three times in PBS before permeabilized by 0.1% Triton X-100. Nonspecific binding sites were blocked with 10% BSA in PBS for 1 h. Primary monoclonal antibodies for iNOS (1:100; Abcam, Cambridge, UK) and CD206 (1:100; Abcam) were incubated in PBS containing 1% BSA at 4°C overnight. Cells were washed three times in PBS. Cells were incubated with goat anti-rabbit Alexa Fluor 488 (1:200) and goat anti-rabbit Alexa Fluor 594 (1:200; Abcam) at room temperature for 2 h as secondary antibodies. Cell nuclei were stained with DAPI for 15 min. The cells were then washed three times in PBS. Images were processed on an LSM5 confocal microscope (Carl Zeiss, Oberkochen, Germany).
6
Intestinal Cell Imaging and Visualization
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Distal small intestine was embedded in O.C.T. compound (VWR chemicals) and cut in 10 µm sections. Sections were stained with 1° antibodies mouse anti-E-cadherin (1.25 µg/ml, clone 36/E-Cadherin, BD Biosciences) and rabbit anti-Ki67 (1/200, clone SP6, Abcam) and 2° antibodies goat anti-mouse Alexa Fluor 488 and goat anti-rabbit Alexa Fluor 594 (4 µg/ml, both from Abcam) and DAPI (1/2,000, Cat No. D1306 Invitrogen). Sections were mounted with ProLong Gold antifade reagent (Molecular Probes). Images were acquired on an LSM 780 confocal microscope (Zeiss) using the 20× magnification objective.
7
Immunofluorescence Assay for γH2A.X
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Briefly, the cells on slides were immobilized using 4% paraformaldehyde and permeabilized using 0.1% Triton X-100 in PBS. Then, the cells were blocked using goat serum (Zhongshan Golden Bridge, China) and incubated overnight at 4°C with anti-γH2A.X antibody (Cell Signaling, MA, USA, 1 : 500). After washing, the cells were incubated with goat anti-rabbit Alexa Fluor 594 (Abcam, UK, 1 : 1000). The cells were then counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Bioworld, China, 1 : 2000) for 5 min. The slides were imaged using a fluorescence microscope (Leica, Germany).
8
Identifying Fos-Expressing Cells in SVZ and AON
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In order to reveal the phenotype of Fos producing cells in the SVZ and AON, brain sections of both control and experimental rats were processed for double immunofluorescent labeling. For recognition of astrocytes and neurons, glial fibrillary acidic protein (GFAP) and neuronal nuclear antigen (NeuN) were used, respectively.
The sections were washed three times in PBS and then incubated in 3% normal goat serum in PBS and 0.25% Triton X-100 for 2 hours. Subsequently, they were incubated with mixture of two antibodies: either polyclonal rabbit anti-c-Fos (1:10 000; Oncogene) and monoclonal mouse anti GFAP (1:500; Merck Millipore) or polyclonal rabbit anti c-Fos (1:10 000; Oncogene) and monoclonal mouse anti NeuN (1:500; Merck Millipore) for 48 hours at 4ºC. After the incubation, the sections were washed three times in PBS and labeled with mixture of corresponding antibodies: goat anti rabbit Alexa Fluor 594 (1:200; Abcam), goat anti mouse Alexa Flour 488 (1:200, Abcam) for 2 hours at room temperature. Then, the sections were washed three times in PBS. After the processing, the sections were mounted on glass slides, airdried and coverslipped with Fluoromont (SERVA).
The sections were washed three times in PBS and then incubated in 3% normal goat serum in PBS and 0.25% Triton X-100 for 2 hours. Subsequently, they were incubated with mixture of two antibodies: either polyclonal rabbit anti-c-Fos (1:10 000; Oncogene) and monoclonal mouse anti GFAP (1:500; Merck Millipore) or polyclonal rabbit anti c-Fos (1:10 000; Oncogene) and monoclonal mouse anti NeuN (1:500; Merck Millipore) for 48 hours at 4ºC. After the incubation, the sections were washed three times in PBS and labeled with mixture of corresponding antibodies: goat anti rabbit Alexa Fluor 594 (1:200; Abcam), goat anti mouse Alexa Flour 488 (1:200, Abcam) for 2 hours at room temperature. Then, the sections were washed three times in PBS. After the processing, the sections were mounted on glass slides, airdried and coverslipped with Fluoromont (SERVA).
9
Hippocampal Immunofluorescence Staining Protocol
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Sections were processed for staining using our previously described immunofluorescence procedure [39 (link)]. Mice were anesthetized and transcardially perfused with 4% paraformaldehyde (PFA). Free-floating, 40-μm-thick coronal sections of the entire hippocampus were collected on a freezing microtome (Leica SM2010R). The primary antibodies were as follows: rabbit anti-PV (1:500; Abcam), chicken anti-Arg1 (1:1000; from Dr. Robert W. Caldwell, Augusta University, USA), rabbit anti-Iba1 (1:500; Abcam) and mouse anti-GFAP (1:1000 Sigma). The secondary antibodies include Alexa Fluor 488 goat anti-rabbit (1:1000; Abcam), FITC goat anti-chicken (1:1000; Invitrogen), Alexa Fluor 594 goat anti-rabbit (1:1000; Abcam) and Alexa Fluor 594 goat anti-mouse (1:1000; Abcam). For details, see SI Methods .
10
Quantitative Analysis of Cav2.1 Expression in Hippocampal and Neocortical Regions
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Immunofluorescence staining and confocal microscopy were used to determine expression of Cav2.1 (Alomone Labs, Ltd, Jerusalem, Israel) (1: 100) and NeuN (Millipore, Billerica, MA, USA) (1:500). The staining procedures were performed as previously described (Ryu et al., 2006 (link)). The specimens were incubated for 60 min with AlexaFluor 594 goat anti-rabbit (abcam, Cambridge, UK) (1:400) and alexafluor 488 goat anti-mouse (abcam, Cambridge, UK) (1:200) after incubation of the primary antibody. Images were analyzed using an A1 Nikon confocal laser scanning microscope (Nikon, Tokyo, Japan). In order to investigate the extent of reduction of Cav2.1 over hippocampal subregions and some neocortex regions, we calculate the intensity (%) of Cav2.1 expression in cKO compared to the control. t-test was performed to compare the group difference.
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