PMC:5118445 / 7590-9664 JSONTXT

{"project":"TEST0","denotations":[{"id":"27920776-65-71-3175570","span":{"begin":1971,"end":1973},"obj":"[\"22743772\"]"},{"id":"27920776-20-26-3175571","span":{"begin":2033,"end":2035},"obj":"[\"23906423\"]"}],"text":"Stepwise Procedures\n\nImmunofluorescence of Tissue Sections\nThe mouse tissue had been fixed in situ by transcardial perfusion with 2% paraformaldehyde solution in TRIS-buffered saline (TBS, pH 7.4). Following this, the lungs were carefully removed and post-fixed in 2% paraformaldehyde for 16–20 h at RT. The tissue was then dehydrated and paraffin-embedded (60°C) using a Leica TP 1020 tissue processor. Human brain fungal abscess tissue was from archived paraffin blocks; fixation conditions are not known.\nParaffin blocks were cut at 3 μm, sections were mounted and dried on Superfrost Plus slides (Thermo Scientific) avoiding temperatures above 37°C. After dewaxing and rehydration, sections were incubated in one of the HIER buffers at different temperatures [20 min at 96°C in a steam cooker (Braun) or 90 min at lower temperatures in a water bath, details in Table 1].\nAfter antigen retrieval, sections were left in the respective HIER buffer at RT to cool below 30°C, rinsed with deionized water three times, TBS pH7.4 one time, and permeabilized for 5 min with 0.5% Triton X100 in TBS at RT, followed by three rinsing steps with TBS.\nSections were surrounded with PAP-pen and treated with blocking buffer for 30 min to prevent non-specific binding. Primary antibodies (Table 1) were diluted in blocking buffer and incubated on the sections over night at 37°C. At any one time, two or three primary antibodies requiring the same antigen retrieval protocol raised in different hosts were combined. We used secondary antibodies raised in donkey and pre-absorbed against serum proteins from multiple host species (Jackson ImmunoResearch). Dilution and blocking buffer was TBS supplemented with 1% BSA/2% donkey NS/5% cold water fish gelatin/0.05% Tween 20/0.05%Triton X100.\n\nHematoxylin/Eosin Histology\nConsecutive sections were stained with hematoxylin/eosin using standard protocols.\n\nImage Analysis\nImage sets were analyzed using the Fiji-ImageJ software package (21) and a common spreadsheet application. The FigureJ plugin (22) was used to assemble Figures 2 and 3."}

{"project":"2_test","denotations":[{"id":"27920776-22743772-34775609","span":{"begin":1971,"end":1973},"obj":"22743772"},{"id":"27920776-23906423-34775610","span":{"begin":2033,"end":2035},"obj":"23906423"}],"text":"Stepwise Procedures\n\nImmunofluorescence of Tissue Sections\nThe mouse tissue had been fixed in situ by transcardial perfusion with 2% paraformaldehyde solution in TRIS-buffered saline (TBS, pH 7.4). Following this, the lungs were carefully removed and post-fixed in 2% paraformaldehyde for 16–20 h at RT. The tissue was then dehydrated and paraffin-embedded (60°C) using a Leica TP 1020 tissue processor. Human brain fungal abscess tissue was from archived paraffin blocks; fixation conditions are not known.\nParaffin blocks were cut at 3 μm, sections were mounted and dried on Superfrost Plus slides (Thermo Scientific) avoiding temperatures above 37°C. After dewaxing and rehydration, sections were incubated in one of the HIER buffers at different temperatures [20 min at 96°C in a steam cooker (Braun) or 90 min at lower temperatures in a water bath, details in Table 1].\nAfter antigen retrieval, sections were left in the respective HIER buffer at RT to cool below 30°C, rinsed with deionized water three times, TBS pH7.4 one time, and permeabilized for 5 min with 0.5% Triton X100 in TBS at RT, followed by three rinsing steps with TBS.\nSections were surrounded with PAP-pen and treated with blocking buffer for 30 min to prevent non-specific binding. Primary antibodies (Table 1) were diluted in blocking buffer and incubated on the sections over night at 37°C. At any one time, two or three primary antibodies requiring the same antigen retrieval protocol raised in different hosts were combined. We used secondary antibodies raised in donkey and pre-absorbed against serum proteins from multiple host species (Jackson ImmunoResearch). Dilution and blocking buffer was TBS supplemented with 1% BSA/2% donkey NS/5% cold water fish gelatin/0.05% Tween 20/0.05%Triton X100.\n\nHematoxylin/Eosin Histology\nConsecutive sections were stained with hematoxylin/eosin using standard protocols.\n\nImage Analysis\nImage sets were analyzed using the Fiji-ImageJ software package (21) and a common spreadsheet application. The FigureJ plugin (22) was used to assemble Figures 2 and 3."}

{"project":"MyTest","denotations":[{"id":"27920776-22743772-34775609","span":{"begin":1971,"end":1973},"obj":"22743772"},{"id":"27920776-23906423-34775610","span":{"begin":2033,"end":2035},"obj":"23906423"}],"namespaces":[{"prefix":"_base","uri":"https://www.uniprot.org/uniprot/testbase"},{"prefix":"UniProtKB","uri":"https://www.uniprot.org/uniprot/"},{"prefix":"uniprot","uri":"https://www.uniprot.org/uniprotkb/"}],"text":"Stepwise Procedures\n\nImmunofluorescence of Tissue Sections\nThe mouse tissue had been fixed in situ by transcardial perfusion with 2% paraformaldehyde solution in TRIS-buffered saline (TBS, pH 7.4). Following this, the lungs were carefully removed and post-fixed in 2% paraformaldehyde for 16–20 h at RT. The tissue was then dehydrated and paraffin-embedded (60°C) using a Leica TP 1020 tissue processor. Human brain fungal abscess tissue was from archived paraffin blocks; fixation conditions are not known.\nParaffin blocks were cut at 3 μm, sections were mounted and dried on Superfrost Plus slides (Thermo Scientific) avoiding temperatures above 37°C. After dewaxing and rehydration, sections were incubated in one of the HIER buffers at different temperatures [20 min at 96°C in a steam cooker (Braun) or 90 min at lower temperatures in a water bath, details in Table 1].\nAfter antigen retrieval, sections were left in the respective HIER buffer at RT to cool below 30°C, rinsed with deionized water three times, TBS pH7.4 one time, and permeabilized for 5 min with 0.5% Triton X100 in TBS at RT, followed by three rinsing steps with TBS.\nSections were surrounded with PAP-pen and treated with blocking buffer for 30 min to prevent non-specific binding. Primary antibodies (Table 1) were diluted in blocking buffer and incubated on the sections over night at 37°C. At any one time, two or three primary antibodies requiring the same antigen retrieval protocol raised in different hosts were combined. We used secondary antibodies raised in donkey and pre-absorbed against serum proteins from multiple host species (Jackson ImmunoResearch). Dilution and blocking buffer was TBS supplemented with 1% BSA/2% donkey NS/5% cold water fish gelatin/0.05% Tween 20/0.05%Triton X100.\n\nHematoxylin/Eosin Histology\nConsecutive sections were stained with hematoxylin/eosin using standard protocols.\n\nImage Analysis\nImage sets were analyzed using the Fiji-ImageJ software package (21) and a common spreadsheet application. The FigureJ plugin (22) was used to assemble Figures 2 and 3."}