Mounted and dried sections were washed 3 × 5 min in PBS before heat-based antigen retrieval was performed to unmask fixed antigens following the manufacturer’s instructions (Vector Laboratories, H-3301). After a 30 min cooling period, slides were washed 3 × 5 min in PBS and incubated in primary antibody diluted in 0.3% Triton-X100/PBS with 4% milk powder over night at room temperature. The following concentrations were used: 1:5 (mix clone supernatants), 1:100 (final clone supernatant), 1:500,000 (7B7-A3, pigeon), 1:500,000 (7B7-A3, zebra finch), 1:5000 (7B7-A3, chicken). The next day, slides were washed 3 × 5 minutes in PBS, incubated with the secondary antibody (1:1000, anti-mouse, Vector Laboratories, PK-6100) for 2 hours at room temperature, washed, and incubated with the Vectastain Elite ABC HRP reagent (Vector Laboratories, PK-6100) for 1 h. After another round of washing, slides were incubated for 1 min in PBS supplemented with 0.06% Diaminobenzidine (Sigma Aldrich, D5905) and 0.08% H2O2. After a final washing step, slides were dehydrated in serial dilutions of ethanol and cover slipped.
H 3301
Manufactured by Vector Laboratories
Sourced in United States
The H-3301 is a versatile laboratory instrument designed for general-purpose analytical applications. It is a compact, benchtop device that offers reliable and consistent performance. The core function of the H-3301 is to provide accurate and reproducible measurements for a variety of sample types.
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Lab products found in correlation
H 3300, by Vector Laboratories (4 mentions)
Novared peroxidase substrate kit, by Vector Laboratories (2 mentions)
Tip ptd p02, by Cosmo Bio (2 mentions)
Dabco, by Merck Group (2 mentions)
Axio slide scanner, by Zeiss (2 mentions)
Axioscan, by Zeiss (2 mentions)
Vectastain elite abc hrp reagent, by Vector Laboratories (1 mentions)
Pk 6100, by Vector Laboratories (1 mentions)
Cellsens software, by Olympus (1 mentions)
D9542, by Merck Group (1 mentions)
Alexa fluor 488 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Pontamine sky blue, by Merck Group (1 mentions)
Alexa fluor 488 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 labeled goat anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ab32575, by Abcam (1 mentions)
Anti α sma, by Merck Group (1 mentions)
A11073, by Thermo Fisher Scientific (1 mentions)
Z0622, by Agilent Technologies (1 mentions)
Bovine serum albumin (bsa), by Jackson ImmunoResearch (1 mentions)
Spotfire, by TIBCO Software (1 mentions)
20 protocols using h 3301
1
Immunohistochemistry Protocol for Avian Tissue
2
Immunohistochemical Analysis of TDP-43 and ESRRG
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On day one, sections were deparaffinized with Citrisolv (FISHER brand #04-355-121) and hydrated through a serial dilution of ethanol. Sections were permeabilized with 1% FBS (Atlanta Biologicals #511150) and 0.2% Triton X-100 (Sigma #65H2616). Following permeabilization, antigen retrieval was performed in a high pH solution (Vector # H- 3301) in a pressure cooker for 20 min at 120 °C. Next, sections were blocked with 2% FBS in 1 × PBS for 60 min and were incubated with primary antibody overnight. Primary antibodies were diluted in 2% FBS in 1X PBS. For TDP-43, we used mouse monoclonal anti-TDP-43 (NovusBiologicals, # H00023435-M01, 1/500) and for ESRRG we used rabbit polyclonal anti-ESRRG (1:500, ProteinTech, Cat# 14017-1-AP). On day two, slides were incubated with secondary antibodies (goat anti-mouse Abcam #1150116, Alexa594, 1/500 and donkey anti-rabbit Jackson Immuno Research #715545, Alexa 488, 1/500) for 60 min at room temperature. Secondary antibodies were diluted in 2% FBS in 1X PBS. We quenched CNS auto-fluorescence with 0.1% Sudan Black in 70% ethanol for 15 s. Slides were cover slipped using ProLong Gold Antifade Mountant with DAPI. We analyzed 2 8 µm sections per patient.
3
Quantifying NF-κB p65 Activation in Murine Livers
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IP LPS-exposed and control livers from p7 and adult mice were fixed with 4% paraformaldehyde, paraffin-embedded, and stained against NF-κB p65 subunit. Briefly, after antigen retrieval (antigen unmasking solution, H-3301, Vector Laboratories, Burlingame, CA, USA), permeabilization (0.5% Triton X), and quenching with 100 mM glycine and 0.5% Pontamine Sky Blue (Chicago Sky Blue 6B, C8679-25G, Sigma-Aldrich, St. Louis, MO, USA), 5 μm sections were blocked with Sea Blocking (#37527, ThermoFisher Scientific, Waltham, MA) for 40 min, and Fc Receptor Blocking (NB309-15, Innovex Biosciences, Richmond, CA, USA) for 30 min. Sections were subsequently incubated with anti-p65 antibody (1:100, NF-κB p65 XP®, #8242, Cell Signaling, Danvers, MA, USA) at 4°C overnight. The following day, sections were incubated with secondary antibody (1:200, Alexa Fluor 488 donkey anti-rabbit, A-21206, ThermoFisher Scientific, Waltham, MA) for 1 h at room temperature. Finally, sections were mounted with DAPI (1:1000, D9542, Sigma-Aldrich, St. Louis, MO, USA) and imaged with an IX83 microscope and DP80 camera using CellSens software (Olympus Life Science, Waltham, MA, USA).
4
Immunolabeling of Phosphorylated Signaling Proteins
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Embryos and tissue explants were fixed in either 4% PFA or 10% Neutral Buffered Formalin (NBF) for one hour on ice. The samples were embedded and cryosectioned as described. The immunolabeling protocol was the same as the cell shape visualization protocol described above except that the incubation time with the primary antibodies ranged from overnight (pSmad2 and pERK) to four days (pSmad1/5/8) at 4°C. For pERK staining, antigen retrieval was performed by microwaving in Antigen Unmasking solution (H-3301, Vectorlabs). The following antibodies were used: primary antibodies used were pSmad2 (1:25, rabbit polyclonal, 3101, Cell Signaling Technology, RRID:AB_331673), pSmad1/5/8 (1:100, rabbit polyclonal, 9511, Cell Signaling Technology, RRID:AB_331671), pERK (1:5, rabbit polyclonal, 9101, Cell Signaling Technology, RRID:AB_2315113); and the secondary antibody was Rabbit Goat anti-rabbit Alexa-Fluor 488 secondary antibody (1:400, A-11008, Molecular Probes).
5
Immunohistochemical Analysis of TDP-43 Pathology
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Formalin-fixed paraffin-embedded sections with 6 µm thickness were deparaffinized with Citrisolv (Decon Lab #1601H) and hydrated with different dilutions of alcohol. Endogenous peroxidase activity was quenched with 0.06% H2O2 in methanol for 15 min. Antigen retrieval was performed in a high pH solution (Vector #H-3301) in a pressure cooker at 125 °C for 20 min. Sections were blocked with 2% FBS (Gibco #10438–026) and 0.2% Triton X-100 (Sigma #T8787) in PBS for 60 min. Following antigen retrieval and blocking, the sections were incubated with primary antibodies at 4 °C overnight as follows: TDP-43 (anti-Rabbit, Proteintech #10782–2-AP, 1:5,000) and phosphorylated TDP-43 (pTDP-43) (pS409/410, anti-Rabbit, Cosmobio #TIP-PTD-P02, 1:1,000). The secondary antibody (ImmPRESS HRP Reagent Kit, anti-Rabbit IgG, Vector #MP-7401) was incubated at room temperature for 60 min, and signals were detected using NovaRED peroxidase substrate kit (Vector #SK-4800) for 2 minutes. Counterstaining was performed with hematoxylin (Ricca chemical #3537–32). TDP-43 and pTDP-43 immunohistochemistry was performed using consecutive sections for each patient.
6
Immunohistochemical Analysis of HHIPL1 in Aortic Roots
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Serial frozen aortic root sections from Apoe−/− and wild-type mice were stained by immunohistochemistry with anti-HHIPL1 (HPA052767; Atlas Antibodies), MOMA-2, and SMA (ab32575; Abcam) antibodies using the same methodology as before. Immunofluorescence was performed on paraffin-embedded aortic root sections from Apoe−/− mice using anti-HHIPL1 antibody and Cy3-conjugated mouse monoclonal anti–α-SMA (1:200 C6198, Sigma-Aldrich) antibodies. Sections were treated with antigen unmasking solution (H-3301, Vector Laboratories). Alexa Fluor 488–labeled goat anti-mouse secondary antibody (Thermo Fisher) was used to detect anti-HHIPL1 antibody localization. Images were acquired with a DM2500 Leica fluorescent microscope. Dual staining was quantified across 3 sections using ImageJ.
7
SARS-CoV-2 Immunohistochemistry in Lung Tissue
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5μm sections of formalin-fixed, paraffin-embedded lung were mounted on charged glass slides, baked for one hour at 60°C, and passed through Xylene, graded ethanol, and double distilled water to remove paraffin and rehydrate tissue sections. A microwave was used for heat induced epitope retrieval. Slides were heated in a high pH solution (Vector Labs H-3301), rinsed in hot water and transferred to a heated low pH solution (Vector Labs H-3300) where they were allowed to cool to room temperature. Sections were washed in a solution of phosphate-buffered saline and fish gelatin (PBS-FSG) and transferred to a humidified chamber, for staining at room temperature. Tissues were blocked with 10% normal goat serum (NGS) for 40 minutes, followed by a 60-minute incubation with a guinea pig anti-SARS antibody (BEI NR-10361) diluted 1:1000 in NGS. Slides were washed and transferred to the humidified chamber for a 40-minute incubation with a goat anti-guinea pig secondary antibody (Invitrogen A11073) tagged with Alexa Fluor 488 and diluted 1:1000 in NGS. Following washes, DAPI (4’,6-diamidino-2-phenylindole) was used to label the nuclei of each section. Slides were mounted using a homemade anti-quenching mounting media containing Mowiol (Calbiochem#475904) and DABCO (Sigma #D2522) and imaged at 20X with a Zeiss Axio Slide Scanner.
8
Immunostaining of SARS-CoV-2 in Lung Tissue
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Lung tissue sections were fixed in zinc formalin, embedded in paraffin, and then subjected to epitope retrieval using solutions of varying pH (Vector Labs H-3301 and H-3300) as previously described in [26 (link),27 (link)]. Blocking was performed using BSA/or serum, followed by incubation with primary and secondary antibodies. The dilutions and the primary antibodies were 1:1000 anti-SARS (NR-10361, BEI, Manassas, VA, USA), 1:500 anti-Spc (WRAB-9337, Seven Hills Bioreagents, Cincinnati, OH, USA), and 1:20 anti-cytokeratin antibody (Z0622, Dako, Glostrup, Denmark). The corresponding secondary antibodies were added as detailed in [27 (link)]. Digital imaging was executed using a Zeiss Axio scan. Z1. (Jena, Germany).
9
Multiplex Tissue Imaging Analysis
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Imaging analyses were performed using the Opal multiplex tissue staining system and the Mantra quantitative pathology workstation (PerkinElmer, Waltham, MA). Slides from biopsied tumor tissues were stained using an Opal multiplex tissue staining system (PerkinElmer, Waltham, MA). Antigen retrieval was performed by heating slides to 93 ± 2°C for 20 minutes in a high-pH antigen unmasking solution (H-3301, Vector Labs, Burlingame, CA), followed by blocking in 5% bovine serum albumin (BSA) (Jackson ImmunoResearch, Birmingham, AL) in phosphate-buffered saline (PBS). Cell phenotyping and counting were performed using the Mantra quantitative pathology workstation (PerkinElmer) within representative fields preselected by trained hematopathologists. Spatial distribution and marker intensity in target cells were analyzed using inForm® image analysis (PerkinElmer) and Spotfire (TIBCO Software, Palo Alto, CA) software.
10
Immunodetection of HuR in Murine Tail Tissues
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